There are a number of philosophies as to which organisms provide the most in-teresting bioactive metabolites. In the present work, we are focused on bacteria with a taxonomic diversity in order to provide the greatest possible chemical variety. For this purpose, the study was planned to be carried out on two marine bacterial classes living in different habitats in comparison to terrestrial Streptomyces bacteria.
• Marine Bacteria other than Streptomycetes
North Sea bacteria were isolated from the free water column at the island of Helgoland from the German Wadden Sea by I. Wagner-Döbler (Braunschweig) and M. Meiners (Emden). The taxonomy of the non-Streptomycetes was unknown in ad-vance and was determined only if the chemical results would justify it. The collected organisms were therefore described temporarily by colour, morphology, presence of mucus, odor etc. In situ and deckside photographs were important for the later taxo-nomic evaluation and were performed with a digital camera. The non-Streptomycetes were mainly cultivated on LB-medium with artificial sea water, and showed different coloured colonies (including white) on the agar plates, and most likely characterised by an increasing pH value through the fermentation process arriving at ≈ 8.5 at the end. Most of these types of strains produce indole alkaloids.
• Marine Streptomycetes
The marine Streptomycetes were deposited in the Actinomycetes culture collec-tion of the Alfred-Wegner Institute für Polar- und Meeresforschung in Bremerhaven and identified by E. Helmke. They were mainly cultivated on M2+ medium (= M2
medium + sea water). The fermentation process showed mostly a decrease in the pH value (< 5) and increased at the last stages of the cultivation.
• Terrestrial Streptomycetes
The terrestrial Streptomycetes were obtained from the strain collection of bioLeads, Heidelberg. They were also cultivated on M2 medium, however, with tap water. They showed the same fermentation behaviour as in case of marine Strepto-mycetes.
The general working up procedure of the investigated strains can be summarized in the following steps (Figure 3). The strains were evaluated first through chemical and biological screening. The interesting strains were then cultivated at large scale and after isolation of the metabolites, structure elucidations were performed.
3.1.1 Primary screening
Due to the highly different metabolic capabilities of the available bacterial strains, it was essential to select effective strains either for their biological activity, or for their production of new natural products. Hence well-grown 1-L shaker cultures were freeze-dried and the resulting residue was extracted with ethyl acetate (3 times), and evaporated under vacuum at 40 °C.
To evaluate the antibiotic activity of the extracts, they were subjected to agar diffusion tests using Escherichia coli, Streptomyces viridochromogenes (Tü57), Ba-cillus subtilis, Staphylococcus aureus, Mucor miehei (Tü284), Candida albicans, Chlorella vulgaris, Chlorella sorokiniana, and Scenedesmus subspicatus as test or-ganisms. In parallel, the cytotoxic activity was evaluated against brine shrimps (Ar-temia salina) and nematodes (Caenorhabditis elegans). The extracts were also chemically screened by TLC, using UV and spray reagents, as well as by HPLC analysis to dereplicate the known compounds and to avoid the unwanted strains.
Chemical and biological screenings complement each other very well: The sensitiv-ity of biological methods is much higher than that of the chemical analyses and can detect even traces, whereas the chemical screening targets new structures even if they are not obviously bioactive. The strains that produce interesting metabolites are sub-jected to the scale-up cultivation.
Search in AntiBase, DNP and CA
Ingnored Isolated strains
1-l-Shaker culture Storage
1. Freez-drying
2. Extraction by ethyl acetate
Crude extract
Chemical screening
Evaluation of the results
Fermentation on large scale
Biological screening
Isolation and structure elucidation
Provision the bioactivity of the afforded pure compounds Uninteresting strains
Intersting strains
Figure 3: Working up of the selected strains in a general screening.
3.2 Large scale cultivation and extraction
As most strains are producing only metabolite concentrations of 0.1–1 mg/l, fermentation in at least 20∼50 liters scale is necessary to get an adequate amount of product. This is mostly a two-step process: The initial agar culture of the producing organism will be transferred to a 2-liter liquid culture and then scale-up to provide up to 50 liters of culture broth. The fermentation may be carried out in shaking flasks or in a fermenter. It is worth to mention that, most of coloured compounds (e.g.
quinones), are produced better in shaker culture than in a jar fermenter.
acetate and the process continued until no further colour is extracted. Extraction can be considered complete when little or no additional residue is obtained after concen-tration. Storing extracts in ethyl acetate at room temperature can lead to degradation of the compounds and lower overall yields. Solutions should be therefore evaporated as soon as possible, and it is strongly recommended to store the residues at the cold-est temperature possible to minimize degradation of compounds.
Adsorption on XAD resin is another efficient extraction method for obtaining the crude extracts. For this purpose, the culture filtrate is passed at a suitable flow rate through a glass column containing XAD resin (XAD-2). The compounds are eluted from XAD usually with methanol or a methanol/water gradient. Extraction with XAD is more advisable than the commonly used ethyl acetate extraction be-cause of its cheapness. Also highly polar water-soluble compounds can be obtained if lipophilic interactions are possible, good recovery rates are obtained, and it is easy to recover and purify the resin for further use. In contrast to solvents, the resin is not harmful.
The isolation procedures depend mainly on the polarity of the compounds of in-terest (which can be determined by thin layer chromatography with eluents of vary-ing polarity). There are two preliminary separation systems, which are commonly suitable for most metabolites:
• Flash chromatography of the extract on silica gel using a stepwise gradient of dichloromethane/methanol or ethyl acetate/cyclohexane. This system classi-fies the fractions depending on their polarity. Disadvantage is the contact with silica gel, as this may rearrange, oxidise, cleave or even destroy metabo-lites.
• Size-exclusion chromatography using Sephadex LH-20. The separation is based on the molecular weight. Sephadex does not have the former disadvan-tages and the recovery rate for the compounds is also higher.
The afforded fractions are monitored by TLC to decide the next isolation steps which may be by PTLC, silica gel column chromatography, Sephadex LH-20 or HPLC, etc.
3.3 Dereplication concept
It is obvious that despite of the existence of modern methods, the isolation and structural elucidation of natural compounds is a time-consuming and expensive proc-ess. The dereplication is an important step with the aim to distinguish between known compounds and unknowns, and consequently allowing to exclude the known compounds at an earlier stage.
The principle of this method is to compare data fragments of mixtures or pure metabolites with suitable literature data. This might be carried out by comparing the UV[76] or MS data and HPLC retention times with appropriate reference data collec-tions. This method needs only negligible sample amounts and affords reliable results, if authentic samples had been available to measure the reference data. UV data and MS fragmentation patterns are also useful to identify unknown metabolites, if these show similar chromophores or fragmentation patterns as known analogues. Presently, ESI MS/MS spectra of more than 1000 of the most frequently isolated substances are included in our database of natural products. First results have shown that already known natural products can be identified easily even from crude extracts obtained from bacterial broths. Application of these methods is a very valuable tool to make the process of finding new biological and pharmacological active compounds more efficient.
As it will never be possible to collect a complete sample set and to measure all experimental data under identical conditions, reference values from the literature have to be used. If NMR data are selected, results from 1D measurements can be translated into substructures, which then will be used for a database search. In this case, normally sufficiently pure samples are required.
Databases with the NMR or UV data and a variety of other molecular descrip-tors can be searched using computers[77]. The most comprehensive data collection of natural compounds is the Dictionary of Natural Products (DNP)[24], which compiles metabolites from all natural sources, including plants. Our own data collection (An-tiBase[23]) is, however, more appropriate for the dereplication of microbial products, as the identification depending on structural features and spectroscopic data is more comprehensive, faster and more reliable. In the case of new compounds, a database search is also helpful because novel skeletons are rare and usually related compounds
bank of information worldwide, is used for a final confirmation that a given structure is new. Sub-structure searches with small fragments are not possible here for techni-cal reasons.
The combination of liquid chromatography with detection methods such as NMR spectroscopy (HPLC NMR) and tandem mass spectrometry (HPLC-MS/MS) has recently led to new strategies by which biological matrices, e.g., crude plant ex-tracts[78] or extracts from marine organisms[79], are screened to obtain as much infor-mation as possible about known constituents even with a minimum amount of mate-rial. As most compounds of interest are thermally labile, HPLC-ESI MS/MS would be the method of choice to identify known molecules from multi-component mix-tures with high selectivity and sensitivity[80]. The absolute configuration of the pure components can be confirmed by application of circular dichroism (CD) spectros-copy[81]