Microbial consortia and methods for their use

#Sucralose (4,1′,6′-trichloro-4,1′,6′-trideoxygalactosucrose), a high-intensity sweetener made from sucrose, can be used in many food and beverage applications.

#A number of different synthetic routes for the preparation of sucralose have been developed in which the reactive hydroxyl in the 6 position is first blocked with an acyl group to form a sucrose-6-acylate. The sucrose-6-acylate is then chlorinated to replace the hydroxyls at the 4,1′ and 6′ positions to produce 4,1′,6′-trichloro-4,1′,6′-trideoxygalactosucrose-6-acylate (sucralose-6-acylate), followed by hydrolysis to remove the acyl substituent and thereby produce sucralose. Several synthesis routes for formation of the sucrose-6-acylates involve tin-mediated acylation reactions, with illustrative examples being disclosed in U.S. Pat. Nos. 4,950,746; 5,023,329; 5,089,608; 5,034,551; and 5,470,969, all of which are incorporated herein by reference.

#Various chlorinating agents may be used to chlorinate the sucrose-6-acylate, and most commonly a Vilsmeler-type salt, such as Arnold's Reagent, will be used. One suitable chlorination process is disclosed by Walkup et al. (U.S. Pat. No. 4,980,463), in which a tertiary amide, typically N,N-dimethylformamide (“DMF”), is used as the chlorination reaction solvent. After the chlorination is complete, the reaction mixture is neutralized with aqueous alkali to regenerate the hydroxyl groups at positions 2,3,3′, and 4′ of the sucralose-6-acylate, which yields the sucralose-6-acylate in an aqueous solution, accompanied by the tertiary amide solvent and salts resulting from reactions of the chlorination reagent. The sucralose-6-acylate is then deacylated to produce sucralose. One suitable process is taught by Navia et al., U.S. Pat. No. 5,498,709, the entire disclosure of which is incorporated herein by reference.

#FIG. 1. Salt and temperature ramping curves for acclimatizing consortium P-4 to culture conditions comprising 45° C. and 4.5% salt.

#FIG. 2. Biodegradation of total chlorinated carbohydrates in two waste streams produced at different stages of the sucralose production process.

#A. Biodegradation of total chlorinated carbohydrates in waste stream 1 by consortia P-3 and P-4.

#A method is described for acclimatizing microorganisms to degrade chlorinated carbohydrates that may be found in waste streams. This method has been utilized to produce microbial consortia that can be used to degrade chlorinated carbohydrates. These consortia were deposited with the National Center for Agricultural Utilization Research, Northern Regional Research Laboratories, Peoria, Ill. on Jan. 11, 2008, by Dr. Chi-Li Liu for Tate & Lyle as accession numbers NRRL B-50091, NRRL B-50092, NRRL B-50093, NRRL B-50094, NRRL B-50095, NRRL B-50096, NRRL B-50097, and NRRL B-50098, and are described in Example 3. These microbial consortia contain groups of microbial species adapted to co-exist in an environment in which certain parameters, such as food component(s), temperature, salt concentration, or combinations of these parameters provide selective pressure for survival and biodegradation. By employing microbial consortia for biodegradation, the need to chemically de-chlorinate chlorinated carbohydrates may be reduced or even completely avoided.

#In this description “microbial” encompasses microscopic organisms (microbes, microorganisms) such as bacteria and unicellular eukaryotic organisms. Microbial “growth” is defined as survival or any reproduction of the microbes. A “consortium” is a group of two or more microbial species present in a single culture. A “culture” refers to a single bioreactor containing a growth medium and two or more groups of microbial organisms. A “waste stream” is a medium containing compounds that are generated in a production process, wherein these compounds can not be re-cycled back to the production process or recovered as a useful product.

#Environmental samples containing microorganisms for acclimatization may be obtained from sites of production where chlorinated carbohydrates are likely to be present, for example, waste solutions, waste deposits such as sludge and sediments, and soil in the vicinity of a production site. The environmental sample containing microorganisms is enriched and acclimatized for desired performance by culturing in a synthetic medium which mimics the composition and temperature of the waste stream. Generally these conditions include from 0% to 4.5% by weight chloride salt, more typically from 1% to 4.5%, and a temperature between 15° C. and 55° C. The synthetic medium contains a food source for the microorganisms, such as yeast extract (source of nitrogen, amino acids and vitamins) and sugar (carbon source), as well as inorganic salts (for example calcium, sodium, phosphate, sulfate, etc.) and trace elements (such as Fe, Mn, Ni, Mo, Co, Se, etc.) which are important for the growth of microbes. Survival and growth of different microorganisms present in the sample will depend on the initial food source, concentration of chloride salt, and temperature conditions. In one embodiment, the microorganism cultures are incubated at a temperature between about 27° C. to about 50° C. and the medium has a final chloride salt concentration of about 4.5% or less. The salt content may be composed of any chloride salt or combination of chloride salts, including alkali metals, alkaline earth metals, and transition metals, e.g., NaCl, CaCl2, MgCl2, MnCl2, etc., and ammonium chloride. The pH of the medium may be adjusted to optimize conditions for biodegradation of waste streams.