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Compatible solutes are best described as organic osmolytes responsible for osmotic balance and at the same time compatible with the cells' metabolism. A comprehensive survey (using HPLC and NMR methods) on halophilic/halotolerant eubacteria has revealed the full diversity of compatible solutes employed in nature. Molecular principles derived from the spectrum of compounds found in the bacterial world may be summarized as follows. Compatible solutes are polar, highly soluble molecules and uncharged at physiological pH. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. Using nearinfrared spectroscopy we have also been able to demonstrate that compatible solutes are strong water-structure formers and as such probably excluded from the hydration shell of proteins. This “preferential exclusion” probably explains their function as effective stabilizers of the hydration shell of native proteins (protection against heating freezing and drying). Hence these typical products of halophilic eubacteria have a considerable potential as stabilizing/protecting agents on both molecular and whole-cell level. Thorough understanding of common structural principles and fundamental water-solute interactions will ultimately enable us to design novel highly efficient stress protectants and stabilizers of biomolecules.

A novel biotechnological process called "bacterial milking" has been established for the production of compatible solutes using the Gram-negative bacterium Halomonas elongata. Following a high-cell-density fermentation which provided biomass up to 48 g cell dry weight per liter, we applied alternating osmotic shocks in combination with crossflow filtration techniques to harvest the compatible solutes ectoine and hydroxyectoine. H. elongata, like other halophilic or halotolerant microorganisms, produces compatible solutes in response to the salinity of the medium. When transferred to a low salinity medium (osmotic downshock), H. elongata cells rapidly released their solutes to achieve osmotic equilibrium. Subsequent reincubation in a medium of higher salt concentration resulted in resynthesis of these compatible solutes and-after a defined regeneration time-the procedure could be repeated. By repeatedly performing this "bacterial milking" process (at least nine times) we were able to produce large amounts of ectoines with a biomass productivity of 155 mg of ectoine per cycle per gram cell dry weight. Further purification of the products was achieved by a simple two-step procedure based on cation exchange chromatography and crystallization. The principles described in this article may also be useful for the production of other low-molecular-weight compounds.

In the genus Azospirillum tolerance towards high concentrations of sodium chloride, sucrose or polyethylene glycol increased in the order A. amazonense A. lipoferum A. brasilense and A. halopraeferens. In A. brasilense and A. halopraeferens the compatible solutes trehaloseglutamate and an unknown compound were identified. A. halopraeferens only could convert choline to the potent compatible solute glycine betaine. Acetobacter diazotrophicus tolerated high concentrations of sucrose and polyethylene glycol, but was very sensitive towards sodium chloride. In contrast to the more osmotolerant Azospirillum spp. amino acids such as glutamate, serine and histidine were efficiently utilized as carbon and nitrogen sources and betaine, choline and proline did not relieve osmotic stress. New halotolerant bacteria (strains BE and TC) were isolated from the rhizosphere of rice growing in alkaline, saline soil in India. They were oxidase-positive, Gram-negative, very motile bacteria, which showed pleomorphic growth. In semisolid nitrogen free mineral medium they grew and fixed nitrogen microaerobically. These isolates required sodium ions for growth and they tolerated up to 2 M sodium chloride in nitrogen containing mineral medium. At osmotic stress conditions the efficient compatible solute ectoine was synthesized.

Concentrated salt solutions (brines) occur widely in the natural form of coastal lagoons, salt or soda lakes as well as in the man-made form of salterns or saltworks. They are inhabited by a limited number of specialized microorganisms, which use different strategies of haloadaptation. Extremely halophilic archaebacteria (Halobacteriaceae) compensate the high osmotic pressure of brines by high cytoplasmic K+ion concentrations. This requires appropriate adaptations of both the intracellular and extracellular functional macromolecules. Only some of the halophilic, halotolerant and extremely halophilic eubacteria follow this strategy; while the majority of them, and the halophilic algae, synthesize and accumulate in their cells organic compatible solutes (mono-, disaccharides, glycosylglycerols, sugar alcohols, amino acids, betaines). These compounds have besides their water-binding activity, protective functions for enzymes. In eubacteria the most important compatible solute is glycine betaine. Besides this substance, further new substances have been found, such as ectoine, for which functions as well as biosynthetic pathways have yet to be elucidated.