This letter is to provide you with background information on the inherent safety of ALKEN CLEAR-FLO®, ALKEN ENZ-ODOR®, and other ALKEN® microbial products.

All active strains of bacteria produced are environmental isolates that have been naturally enhanced, but have NOT undergone genetic engineering. These strains are carefully selected for their natural talents as probiotics for aquaculture and plant health; bioremediation of natural water bodies, waste water, petroleum spills, sludge, manure and compost. Our bacterial strains are from the following genera: Acinetobacter, Bacillus, Brevibacillus, Enterobacter, Marinobacter, Nitrosomonas, Nitrobacter, Paenibacillus, Paracoccus, Pseudomonas, Rhodococcus, Sphingomonas, Sporosarcina, Starkeya, Streptomyces and Thiobacillus.

All active strains of fungi and yeast are environmental isolates that have been naturally enhanced, but have NOT undergone genetic engineering. Our fungal strains are from the following genera: Aspergillus, Phanerochaete and Trichoderma. We also use the yeast Saccharomyces cerevesiae.

Strain identification is assured by visual screening, growth on selective media, biochemical reactions (including Biolog, API 50CH, API 20E, API 20NE and API ZYM), Fatty Acid Methyl Ester (FAME) with results compared with the Sherlock database and 16S rRNA analysis with results compared to the MicroSeq database, delivering the most precise identification possible with current state of the art technology. Pure cultures of many strains are stored at the American Type Culture Collection (ATCC) for safekeeping, while other strains are deposited in compliance with the Budapest treaty at the Agricultural Research Service Culture Collection (NRRL), in conjunction with patent applications. The strains of bacteria, yeast and fungi used in Alken-Murray formulas are generally rated as Biosafety Class 1 organisms. Full details of individual strains, identified by "Alken-Murray identification codes", including morphology, source, gram-stain, 16S rRNA, API, Biolog and/or FAME identification, enzyme profile, carbohydrate utilization, distinguishing performance characteristics and antibiotic sensitivity are available to qualified health, agriculture, or customs authorities, with a signed confidentiality agreement, when required for importation of microbial products.

The production process involves subjecting the culture media ingredients, substrates, and the fermentation equipment to temperatures of 121°C for at least one hour before use as a growth medium. Equipment not subjected to heat treatment is sanitized using standard techniques such as acid cleaning and/or chlorination. The sanitizing procedures used all but rule out the possibility of contamination of the product by pathogenic or other undesirable organisms.

The microbes are cultured, individually, in an aqueous medium that contains nutrients such as corn syrup solids, digests of soybean or skim milk, yeast extract, peptones, glucose or other sugars, mineral salts, etc. from initial culturing in successively larger shake-flasks to 5 liter spinner flasks, culminating with submerged fermentation in 5 gallon bioreactors. From there, the process is modified, according to gram-stain and desired end product - liquid or dry.

Gram-negative strains and a few non-sporulating gram-positive strains used in dry products are grown through the 5 gallon bioreactor stage, previously described, and are then poured out onto a sterilized blend of wheat bran, and hulls of rice and soy, for final growth and drying in a process called solid state fermentation, as illustrated in our pictorial factory tour. During culturing, temperature is maintained at 31 °C, while humidity is maintained at 90%, with sterile air exchanged twice per minute. Following the culturing period of 24 to 36 hours, the temperature is increased to between 40° and 43°C, depending on the bacterial strain. Steam filled radiators on each side begin to heat the air being circulated, so that the bran begins to dry. The dried bran in solid cake form, is dumped from the drying trays into a sterilized accumulating hopper, which has an auger that drives it to a hammer mill which breaks the bran into particles small enough to pass through a #10 mesh screen. Pneumatic ducts transfer the ground bran to individual storage bins, labeled by strain name and lot number. Samples are taken from each bin and checked for concentration and purity. Should the concentration not meet its specification, the caked bran is placed in a blender, where more bacteria from a higher count batch of the same strain is added, until specifications are met. The blend is then assigned a new lot number, with notes in the computer about which lot numbers make up the new blended lot.

Gram-negative strains used in liquid products are also grown to the 5 gallon stage, as described above, but they are then transferred to larger bioreactors, always using the submerged fermentation process. After growth for 24 to 36 hours at 30° to 36°C in the final large bioreactor, the liquid is processed through ultra-filtration to increase concentration. The batch is then drummed off and each drum is labeled with the lot number, strain name and date of production. Samples are then withdrawn for QC testing for purity and concentration.

Product specifications, including QC tests ordered, are loaded into the formulary program, which automatically calculates weights of each strain to be added to a formula to achieve the balance and total count called for in the formula, based on actual count of stored strains called for in the batch size ordered for the specific formula, so that the completed product will exceed our guaranteed minimum total and spore count, while maintaining the proper balance of strains to each other in the formula ordered to fill one week's orders for that product. We also allow a bit of overage for products often ordered to solve emergency problems, such as nitrifiers and aquaculture probiotics, so that small rush orders can be filled, while the balance of a distributor's order is processed normally.

Whichever manufacturing process is used (solid state growth on wheat bran, spray-drying, freeze-drying or submerged liquid fermentation), each strain produced and all final products are tested for Salmonella using BD's CHROMagar Salmonella, approved by the AOAC, and for Shigella via the procedure recommended by the United States Department of Agriculture and Standard Methods for the Examination of Water and Wastewater Treatment (18th edition). Other quality control tests that may be included involve performing a standard bacterial plate count (total and/or spore count), bacterial plate counts on other selective media, such as CHROMagar E.coli or 0157 for E.coli, CHROMagar Orientation, MacConkey Agar, Pseudomonas Isolation Agar, etc. depending on the specific formula and strains that should or should not be included. pH and color-matching are also conducted to verify product quality and consistency.

The formulary computer program keeps track of the inventory of each strain in stock and writes a work order for preparation of a new growth cycle when the inventory drops below the quantity needed to meet expected demand, or, in the case of rarely used strains, when the inventoried strain exceeds the age necessary to produce finished products with the shelf-life we guarantee for the recipes that require rarely used strains.

After recipe blending and QC testing are completed, a MANUFACTURER'S CERTIFICATE FORM is issued for each order of Alken Clear-Flo®, Alken Enz-Odor®, Alken Treat-A-Loo and Alken Nu-Bind verifying results of quality control tests for EACH LOT of product ordered. This document, an MSDS and product technical bulletin for each product ordered,, bill of lading, commercial invoice (3 signed copies for export) and any other shipping papers required (SED, NAFTA, Chamber of Commerce authenticated certificate of origin, certificate of non-hazardous cargo for export air shipment, etc. ), are then assembled and attached to the client's order, whether palletized or shipping as single pails or drums.Once all paperwork and shrink wrapping is completed and checked, the freight carrier or client's forwarder is contacted to arrange pickup. If client requests tracking details at the time they place their order, this information will be conveyed by e-mail, fax or telephone, as requested.

Few of our competitors offer a manufacturer's certificate for EVERY batch of EVERY mcrobial product they manufacture.

  Yours truly,

Valerie Anne Edwards

President & CEO

Alken-Murray Corporation

Updated 10/31/2006