The use of salicylaldehyde derivatives as a nitrogen source for antibiotic production by Streptomyces hygroscopicus CH-7

In the present work, four derivatives of salicylaldehyde (salicylaldehyde-hydrazone, phenylhydrazone, semicarbazone and thiosemicarbazone) were synthesized using both conventional (95% ethanol) and green (crude glycerol from biodiesel production) solvents. The obtained compounds were identified by elemental microanalysis, as well as FTIR, UV/Vis and H-NMR spectroscopic methods. Yields of 93–98% of the compounds in crude glycerol were achieved within 10–25 min. The derivatives of salicylaldehyde and crude glycerol were used as a nitrogen and carbon source, respectively, in the medium for antibiotic (hexaene H-85 and azalomycine B) production by Streptomyces hygroscopicus CH-7. The highest concentrations of hexaene H-85 and azalomycine B were achieved in the medium containing salicylaldehyde-thiosemicarbazone (198 and 69 μg/cm, respectively). Derivatives of salicylaldehyde also impacted the strain morphology. In the media with salicylaldehyde-phenylhydrazone and salicylaldehyde-thiosemicarbazone, S. hygroscopicus CH-7 grew like large dispersive pellets with long twisted filaments that produced the highest yield of the antibiotics.

The rapid growth of biodiesel industry has also expanded crude glycerol production as its main by--product [4].The world production of glycerol is predicted to be 41.9 billion L by 2020 [3,5].The increased crude glycerol production will make an additional pressure to already saturated glycerol market and generate environmental concerns associated with contaminated glycerol disposal [2].Therefore, valorization of crude glycerol obtained during biodiesel production through new applications, despite the presence of impurities, has become an important issue for both glycerol market and commercial biodiesel production [2,3].With such an approach, crude glycerol could be regarded as a desirable by-product and not as a waste with a disposal cost.
Crude glycerol originating from biodiesel production has already been used as a solvent in various processes such as Heck coupling and Suzuki cross coupling of halobenzenes [6], base-catalyzed aldol condensation, palladium catalyzed Heck carbon-carbon coupling [2], the Aza-Michael reaction of p-anisidine and the Michael reaction of indole [7].Some Schiff bases can be also obtained in crude glycerol, such as vanillin-semicarbazone.The yield of vanillin-semicarbazone is increased by 17%, and the reaction time is shortened as compared with the use of conventional solvents, such as methanol and ethanol [8].Besides the uses of Schiff bases in medicine and pharmaceutical industry due to their beneficial properties including antibacterial, antifungal and antitumor activities [9], they can be also used as a nitrogen source in antibiotic production by some microorganisms, such as S. hygroscopicus.Previous studies have shown that isatin Schiff base and its inclusion complexes can significantly improve the production of hexaene H-85 and azalomycine B by S. hygroscopicus CH-7 [10][11][12].
In the present work, several derivatives of salicylaldehyde (salicylaldehyde-hydrazone, salicylaldehyde--phenylhydrazone, salicylaldehyde-semicarbazone and salicylaldehyde-thiosemicarbazone) were synthesized in crude glycerol that originated from the alkali-catalyzed transesterification of edible sunflower oil as well as in ethanol (95 vol.%) as a conventional solvent The main goal was to increase the product yield and to shorten the reaction time, as well as to evaluate the obtained derivatives as a nitrogen source in the nutrition medium in antibiotic production by S. hygroscopicus CH-7.Finally, the work aims at increasing possibilities for utilization of crude glycerol in microbiological synthesis of value-added products.

Chemistry
All chemicals used, except crude glycerol, were of reagent grade and used without further purification.They were purchased from Sigma Aldrich.The crude glycerol (impurities < 5%), a by-product in the laboratory production of biodiesel from sunflower oil, was obtained from the Laboratory for Chemical Engineering, the Faculty of Technology, Leskovac.For the purpose of synthesis, the potential excess of methanol was removed from the crude glycerol by distillation.The acidity of methanol-free crude glycerol was adjusted to pH 5 by addition of 85% phosphoric acid [13].The inorganic salts formed in this stage were then removed by centrifugation (6080 RCF for 15 min).
Carbon, hydrogen and nitrogen were determined by microanalysis using a microanalyzer (model 1106, Carlo Erba, Devon, United Kingdom).Melting points were determined in capillary melting point apparatus (model Thomas-Hoover Uni-melt, USA) and were not corrected.The purity of the synthesized compounds was gained by thin layer chromatography (TLC) on silica gel using benzene:chloroform = 55:45, the compound being visualized by iodine vapors.The FTIR spectra were recorded with a spectrometer (Bomem MB-100, model Hartmann & Braun, Canada), using the KBr pellet (1 mg/100 mg) technique.The UV/Vis spectrum of 1×10 -5 mol•dm -3 ethanol solution of synthesized compounds were recorded on a UV/Vis spectrophotometer (Lambda 15, Perkin-Elmer, USA).
Synthesis of salicylaldehyde derivatives was conducted by the standard procedure [14].Salicylaldehyde (0.1 mol) and hydrazine, phenylhydrazine, semicarbazide or thiosemicarbazide (0.1 mol) were dissolved in 95% ethanol as the conventional solvent.The mixture was refluxed at 50 °C and pH was adjusted to 4.5 by adding H 2 SO 4 .The products were collected by filtration and washed out with the used solvent.The experiments were repeated three times.
Salicylaldehyde derivatives were also synthesized in crude glycerol by using the same starting components and molar ratios as stated above.The mixtures were refluxed at 80 °C.The products, precipitated as a solid (salicylaldehyde-hydrazone, salicylaldehyde-phenylhydrazone and salicylaldehyde-semicarbazone) and as a crystal (salicylaldehyde-thiosemicarbazone), were filtered and washed with water.The experiments were repeated three times.

Determination of antibiotics
Antibiotics were measured spectrophotometrically (hexaene H-85 at λ max = 364 nm and azalomycine B at λ max = 252 nm) using a Perkin-Elmer Lambda 15 UV/Vis spectrophotometer (country).The mixture of 0.5 cm 3 fermentation broth and 2.0 cm 3 of 1-butanol for hexaene H-85 and ethyl acetate for szalomycine B was stirred and centrifuged.
The microbial growth was monitored by measuring dry biomass gravimetrically [16].The broth (8 cm 3 ) was centrifuged at 4000 rpm for 15 min to separate the mycelial biomass.After that, the biomass was dried at 105 °C to the constant weight.During the fermentation, samples were periodically taken and microscopic photographs were obtained by Leica (Microsystem, Heerbrugg, Germany) using the Leica application Suite program (ver.2.5.0,Leica microsystem, Switzerland).All experiments were performed in triplicate.

Characterization of salicylaldehyde derivatives
The stoichiometric equation of synthesis is shown in Figure 1.
The synthesized compounds were characterized by physical and spectral studies (given below).Purity of the compounds was checked with TLC, and the melting points are in accordance with literature data [17,18].
Formation of salicylaldehyde derivatives in ethanol, as a conventional solvent, requires an acid-catalyzed reaction (sulfuric acid solution) because of water elimination and nucleophilic attack of the amine component on the salicylaldehyde carbonyl group (pH 4--4.5).The reaction takes place at 50 °C within 30 min (SalH, SalP and SalT) or 40 min (SalS), Table 1.
In crude glycerol, the reaction occurred without the use of catalyst at 80 °C, and compared to the conventional solvent, the reaction times were shorter and the yields of salicyladehyde derivatives were higher (∆ = 2-8%), Table 1.
Among others, the advantages of glycerol, compared to the conventional solvent ethanol, are higher boiling point (290 vs. 78 °C), lower vapor pressure (<133.32 vs. 27997.69Pa) and lower toxicity (lethal dose, 50% (LD 50 ) of 12.600 vs. LD 50 of 5.628), which makes glycerol more suitable solvent for the synthesis.

Antibiotic production with salicylaldehyde derivatives in medium as a nitrogen source
The strain S. hygroscopicus CH-7 produces antibiotics such as hexaene H-85 and azalomycine B. Hexaene H-85 complex produced by Streptomyces CH-7 belongs to polyene antibiotic family.Although not fully characterized, it is structurally similar to mediomycins, linear polyenes consisting of a hexaene moiety and a conjugated oxo-triene group [19].Azalomycine B (elaiophyline) is an antibacterial antibiotic with macrodiolide structure [20].
Polyene antibiotics are potent broad-spectrum antifungal compounds with low frequency of resistance among fungal pathogens.Well-known representatives of this group are nystatin A1 and amphotericin B used in clinical practice as antifungal and antiparasitic drugs.Even though new potential applications of polyene antibiotics in therapy of HIV infection and cancer are proposed, their use is limited due to serious side effects such as nephrotoxicity [21].
Both polyene and macrodiolide antibiotics are synthesized by polyketide syntheses (PKSs).Recently, characterization of modular PKSs responsible for the production of polyketides similar to mediomycins was reported [22].Progress in genomic sequencing and understanding gene cluster evolution as well as chemical studies may significantly contribute to the improved production of known compounds and the discovery of new [23,24].
L-Tryptophan (Trp), as amino acid, has already been used as a nitrogen source, as well as some Schiff bases with isatin moiety, and the results were significant, considering antibiotic production by S. hygroscopicus CH-7 [10,11].In this work, Trp was used as a nitrogen source in the basal medium, while in the modified media it was replaced with an equal amount of SalH, SalP, SalS or SalT.Table 2 shows the impact of basal and modified media on the concentrations of dry biomass and antibiotics, while the kinetics of fermentation is shown in Figure 2. Being very low, the error bars are shown but hardly visible due to low values (±2%).The concentration of dry biomass (Figure 2a) increases within 3 days of fermentation.The highest concentration of dry biomass was achieved in the medium with SalT as a nitrogen source after 2 days (9.2 g/dm 3 ).
The results show that used nitrogen sources have different effects on antibiotic production by S. hygroscopicus CH-7 (Table 2 and Figure 2b and c).
The concentration of hexaene H-85 (Figure 2b) increases within 3 days, while the concentration of azalomycine B (Figure 2c) increases within 5 days in all tested media.The highest concentration of antibiotics is achieved with SalT as a nitrogen source (198 μg/cm 3 for hexaene H-85 and 69 μg/cm 3 for azalomycine B).Concentrations of hexaene H-85 and azalomycine B are higher for 77 and 28%, respectively, than those in the basal medium.The obtained results indicate that medium containing L-tryptophan and Schiff base as a nitrogen source stimulates production of both antibiotics by S. hygroscopicus CH-7.

Effects of salicylaldehyde derivatives on the strain morphology
Composition of media has a great impact on the Streptomyces morphology.The strain used in the present study grows in the form of both single or branched filaments and single pellets.Formation of singles mall pellets, as it was reported, favors the production of secondary metabolites [25,26].The Streptomyces morphology depends on the nitrogen source in the nutrition media.For instance, a strain of Streptomyces fradiae grows like pellets if the nutrition medium contains amino acids or inorganic ammonia salts as nitrogen sources [27].If the nutrition medium contains soybean and inorganic ammonia salts as nitrogen sources, the strain Streptomyces VITSVK9 grows like long and twisted filaments [28].The S. hygroscopicus CH-7 grows like filaments and pellets in the nutrition media containing different amino acids (phenylalanine, tryptophan, alalin, valine and arginine).During the fermentation with Schiff base as a nitrogen source, pellets and single filaments are dominant forms [29].Figure 3 shows the morphology of S.hygroscopicus CH-7 after 7 days.In the basal medium, the strain grows like short and twisted filaments (Figure 4a) while in modified media it is in the form of short filaments (Figure 4b), branched and twisted filaments (Figure 4c) and large dispersive pellets with long twisted filaments.The highest yield of antibiotics was achieved in media with large dispersive pellets with long twisted filaments, with SalT and SalP as a nitrogen source.The results are in accordance with some previous work where isatin derivatives were used as a nitrogen source for antibiotic production.The highest production was also achieved in media where S. hygroscopicus CH-7 grows as pellets with branched filaments [11,22].

CONCLUSION
Synthesis of derivatives of salicylaldehyde were carried out using carbonyl-amine condensation reaction of salicylaldehyde and amines (hydrazine, phenylhydrazine, semicarbazide and thiosemicarbazide) in the stan-  dard solvent 95% ethanol, and in crude glycerol, a green solvent obtained as a byproduct of the biodiesel production.The use of crude glycerol had significant advantages over the conventional solvent since the reaction time was reduced, the yield of the reaction was increased by 15-23% while the utilization of a catalyst was omitted.The synthesized compounds were shown to be good nitrogen sources in medium for production of antibiotics (hexaene H-85 and azalomycine B) by S. hygroscopicus CH-7.The highest concentrations of hexaene H-85 and azalomycine B were achieved in the medium with SalT (198 μg/cm 3 and 69 µg/cm 3 , respectively).Derivatives of salicylaldehyde had also affected the strain morphology.The results have shown that S. hygroscopicus CH-7 grows like large dispersive pellets with long twisted filaments in media with SalT and SalP, in which also the highest yields of antibiotics were achieved.
The present study proves the utility of crude glycerol for application as a solvent in chemical syntheses of value-added products (Schiff bases) that can further improve the microbial synthesis of valuable antibiotics.In addition, the study has demonstrated that crude glycerol can be used as a carbon source in nutrition media for antibiotic production.In line with this, through the valorization of crude glycerol utilization, the present study might contribute to both economic and ecological issues like sustainability of biodiesel production and stabilization of glycerol market.In addition, by using crude glycerol, a problem of toxic solvents in chemical reactions, including syntheses of biological compounds, can be successfully resolved.High boiling point of glycerol makes it useful for higher reaction temperatures and also may lead to higher yields and reaction selectivity as well as shorter times.

Figure 1 .
Figure 1.Reaction scheme of salicylaldehyde derivatives synthesis (a) and structure of tryptophan (b).

Table 1 .
Reaction times and yields of salicylaldehyde derivatives synthesized in crude glycerol and ethanol; Δt -difference between reaction times in ethanol and glycerol; ∆ -difference between reaction yields in ethanol and glycerol

Table 2 .
Effects of tryptophan and derivatives of salicylaldehyde on the maximum dry biomass concentration (X max ) and the maximum concentration of antibiotics (C max ) achieved during the fermentation using S. hygroscopicus CH-7