Alkaline hydrolysis is a simple, natural process by which complex molecules are broken down into their constituent building blocks by the insertion of ions of water (H2O), H+, and OH- between the atoms of the bonds that held those building bocks together. The process occurs in nature when animal tissues and carcasses are buried in soil of neutral or alkaline pH. In this case, alkaline hydrolysis is aided by the digestive processes of soil organisms. Alkaline hydrolysis also occurs in our small intestines after we eat; the complex molecules of proteins, fats, and nucleic acids are hydrolyzed with the aid of digestive enzymes that function most efficiently at a slightly alkaline pH (~pH8.0 to 8.5). Historically, alkaline hydrolysis has been used to study the chemical structure of biological molecules, to prepare skeletal remains for study, and make soaps from animal fats by cooking the fat with lye to release the fatty acids, then cooling the mixture to precipitate the fatty acids as their sodium salts.
Alkaline hydrolysis as an improved alternative to incineration for disposing of waste biologic tissues and animal carcasses is based on the same chemical reaction, with strong alkali and heat used to speed the process.
Chemistry of the Process
Hydrolysis can be catalyzed by enzymes, metal salts, acids, or bases. Bases are typically water solutions of alkali metal hydroxides such as sodium hydroxide (NaOH) or potassium hydroxide (KOH). Heating the reactants dramatically accelerates hydrolysis. Just as proteins, nucleic acids, polymeric carbohydrates, and lipids were made by organisms via the condensation of building blocks, so can they be depolymerized, or unmade, by hydrolysis.
To form peptides and proteins, amino acids are linked to each other in a peptide (amide) bond in which the carboxyl group of one amino acid is condensed to the amino group of another amino acid with the elimination of water. All polypeptides consist primarily of the elements carbon, hydrogen, nitrogen, and oxygen, along with smaller amounts of other elements, mainly sulfur and phosphorous. Hydrolysis reverses the condensation of amino acids into proteins by the acid- or alkali-catalyzed breaking of the peptide bonds and the addition of water at the break. Alkali, in the form of either sodium or potassium hydroxide solution, or a mixture of both, is used at temperatures ranging from ~100ƒC to 180ƒC and higher for rapid dissolution and then hydrolysis of the proteins into small peptides and amino acids in the form of their sodium or potassium salts. Potassium hydroxide or mixtures of potassium hydroxide and sodium hydroxide are the preferred alkali solutions because of the instability of concentrated (50%) stock solutions of NaOH solutions at temperatures below 20ƒC. All proteins, regardless of their origin, are destroyed by alkaline hydrolysis. An example of the chemical composition of animals is shown in Table 1.
Effects of Alkaline Hydrolysis On:
Alkaline hydrolysis leads to the random breaking of nearly 40% of all peptide bonds in proteins, the major solid constituent of animal cells and tissues. The vast majority of the products of the hydrolysis are single amino acids or small peptides in the 2-5 residue range (nearly 98% of the hydrolyzate). Analysis of the hydrolyzate of sheep carcasses digested at a rendering plant in the United Kingdom and subjected to matrix-assisted laser desorption/-ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis showed that the largest peptides found had a molecular weight between 800 and 1,100 Daltons (Da), i.e., were in the range of 7-9 amino acid residues. MALDI-TOF MS is a relatively novel technique in which a co-precipitate of a UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. The ionized biomolecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. The method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides, and oligonucleotides, with molecular masses between 400 and 350,000 Da. It is a very sensitive method, which allows the detection of low (10-15 to 10-18 mole) quantities of sample with an accuracy of 0.1 - 0.01 %. Alkaline hydrolysis generates sodium and/or potassium salts of free amino acids; oligopeptides (small chains of amino acids) are generated as intermediates in the reaction. Some amino acids, such as arginine, asparagine, glutamine, and serine, are destroyed, while others are racemized; i.e., the molecules are structurally modified from a left-handed configuration to a mixture of left-handed and right-handed molecules. In addition, the carbohydrate (sugar) side chains are released from glycoproteins. Under the extreme conditions of temperature and alkali concentration used in the alkaline hydrolysis process, the protein coats of viruses are destroyed and the peptide bonds of prions are broken.
Simple fats consist of three fatty acid chains bound through ester bonds to a molecule of glycerol. During alkaline hydrolysis, all of these ester bonds, as well as the sterol esters and phospholipids of cell secretions and cell membranes, hydrolyze with the consumption of the alkali, producing the sodium and potassium salts of fatty acids, namely soaps. Again, KOH is the preferred alkali because potassium soaps remain liquid as the hydrolyzate cools toward room temperature. Amide groups in glycolipids, another cell membrane constituent, are also hydrolyzed, with consumption of the alkali. Polyunsaturated fatty acids and carotenoids (pigments) undergo molecular rearrangements and are thus destroyed.
As a group of polymers, carbohydrates are the constituents of cells and tissues most slowly affected by alkaline hydrolysis. Both glycogen, the most common large polymer of glucose in animals, and starch, the most common large polymer of glucose in plants, are immediately solubilized. However, the breakdown of these polymers requires much longer treatment than is required for large intracellular and extracellular polymers. Some large carbohydrate molecules, the þ1-4)-linked glycans, such as cellulose, are quite resistant to alkaline hydrolysis, as they are to digestion in the human intestine. On the other hand, cellulosic materials usually occur only in the digestive tracts of grazing animals where, as a rule, they have been macerated and partially digested. Consequently, further degradation, even if slow, usually does not pose a problem. Alkaline hydrolysis also removes critical groups from the molecules of glycoproteins, glycosaminoglycans, and glycolipids, the principal carbohydrates of connective tissue, as well as from the chitinous exoskeletons of insects and other invertebrates (e.g., the carapaces of crabs and lobsters); (1-3)-linked glycans, such as chondroitin sulfates, are slowly degraded. All monosaccharides (simple sugars), such as glucose, galactose, and mannose, are rapidly destroyed by the hot aqueous alkaline solution.
Nucleic acids are large, unbranched, linear polymers held together by phosphodiester bonds, which are similar to the simpler ester bonds of fats but include a phosphate group as part of the bond structure. These ester bonds are also hydrolyzed with consumption of the alkali, rapidly destroying ribonucleic acid (RNA) and more slowly destroying deoxyribonucleic acid (DNA).
Cellulose-based items such as paper, string, undigested plant fibers, and wood shavings (bedding) are among other items that may be associated with animal carcasses. They are not digestible by alkaline hydrolysis but do not interfere with the process. The same is true of rubber, most plastics, ceramics, and stainless-steel items such as catheters, needles, clips, and staples. Silk and collagen sutures, which are proteinaceous, are rapidly digested. The indigestible materials are completely sterilized by the alkaline hydrolysis process. After appropriate treatment of any sharps, these items can be disposed of as ordinary waste at a sanitary landfill. After alkaline hydrolysis, the undigested residue of animal tissues, specifically the inorganic (calcium phosphate) component of bones and teeth, constitutes approximately 3% of the original weight of the tissue (less than 2% of the volume) and remains in the basket as bone ìshadows.î It is completely sterile and is easily crushed to a powder (Figure 1) that can be used as a soil additive.
Applications of the Alkaline Hydrolysis Process
In addition to its utility for the disposal of routinely generated animal tissues and carcasses, alkaline hydrolysis is particularly useful for the disposal of many difficult-to-handle biologic and biohazardous wastes.
Low-Level Radioactive Biological Waste
LLRBW poses a particular problem because of the difficulties and costs inherent in its packaging and disposal at an LLRW burial site. Indeed, the disposal of solid LLRBW has become even more difficult and expensive since 1992. There are still only two LLRW burial sites in the United States. One, in Hanford, WA, accepts LLRW only from the Northwest and Rocky Mountain states; the other, in Barnwell, SC, accepts only limited amounts of LLRW from states other than South Carolina, Vermont, and Connecticut (the Atlantic Compact). The cost of shipping and burying each 55-gallon drum containing radioactive carcasses has increased steadily, as have the surcharges and guarantee fees charged by the sites. The cost of disposing of a single kilo of radioactive animal carcass at Barnwell currently is more than $250. The cost is scheduled to increase rapidly with surcharges until Barnwell closes its doors to out-of-compact waste in 2008.
In contrast, alkaline hydrolysis not only converts animal tissues and carcasses from solid LLRBW to an aqueous solution that is suitable for release to a sanitary sewer under 10CFR20 and derivative state regulations, but does so at a cost of $0.07-$0.13 per kilogram.
Aldehyde-Containing Fixatives and Embalming Fluids
The most common fixative used in preparing tissues for histology and histopathology is 10% formalin (4% formaldehyde); that most often used in preparing tissues for electron microscopy is 2% glutaraldehyde. Glutaraldehyde is also widely used in medicine and biomedical research as a disinfectant for instruments and surfaces. The U.S. Environmental Protection Agency classifies both formalin and glutaraldehyde as ìcharacteristic hazardous wastes.î It is well known that aldehyde groups react with amino groups to form Schiff bases, thus involving the reactive group of the aldehyde in a stable chemical bond and rendering it harmless. This reaction has been the basis for several commercially available solutions for the disposal of waste aldehyde.
Alkaline hydrolysis of animal tissues produces a hydrolyzate solution that contains 5% to 6% amino acids and small peptides. Waste formaldehyde or glutaraldehyde may be added to tissue digestors at the beginning of the process or, preferably, through an accessory port after the digestion cycle has been completed and the hydrolyzate has cooled to below boiling temperature. After addition of the aldehyde, the solution may be heated again and circulated for 15 to 90 minutes (depending on the temperature at which the reaction is run) to allow the aldehyde and amino groups to react. The hydrolyzate produced from the digestion of 100lbs of animal tissue will provide sufficient amino groups to dispose of up to 10 gallons of waste formalin.
Conventional embalming fluids usually contain phenol, another characteristic hazardous waste, in addition to aldehydes. In a hot alkaline solution, formaldehyde and phenol react to form a plastic, BakeliteÆ, which is completely insoluble, thus rendering both hazardous wastes harmless and leaving Bakelite ìcrystalsî of molecular dimensions suspended in the hydrolyzate.
Alkaline hydrolysis at elevated temperature destroys all pathogens listed as index organisms by the State and Territorial Association on Alternative Treatment Technologies (see STAATT I [April 1994] and STAATT II [December 1998] reports). These reports call for a system to be able to prove efficacy in the destruction of infectious agents by producing a 6 log 10 reduction in vegetative infectious agents and a 4 log 10 reduction in spore-forming agents. While each state has its own regulations for approving alternative treatment technologies for regulated medical waste, most of them are derived from or equivalent to the STAATT recommendations.
Results from different laboratories have indicated that combined treatment with heat and alkali destroys the infectivity of brain macerates or homogenates containing prions (proteinaceous infectious particles), the agents that cause transmissible spongiform encephalopathies (TSE) such as mad cow disease (BSE), chronic wasting disease (CWD), and Creutzfeldt-Jakob Disease. Indeed, the World Health Organization (WHO) recommends combinations of alkali and heat treatment as the only method known to be completely effective for destroying TSE agent infectivity.
The U.S. Department of Agriculture and the Environmental Protection Agency have both recommended alkaline hydrolysis as one of only two acceptable treatment and disposal methods for animal tissues and carcasses infected with or suspected of containing prions (the other being incineration at >900ƒC. Alkaline hydrolysis tissue digestors are currently being used in both major chronic wasting disease elimination programs of the USDA. Moreover, large-scale treatment and disposal units are currently being developed for disposal of the specified risk material (SRM) that the USDA has defined (revised regulations 9CFR301-9) in response to the first confirmed case of BSE in the US.
Biological Warfare and Bioterrorism Agents
As recently as the mid-1990s, infectious agents and toxins having potential use as biological-warfare agents could be purchased from suppliers of bacterial cultures, stocks, and biochemicals. Consequently, the U.S. Congress passed legislation making it a Federal crime, in some instances punishable by death, to possess or transport certain agents without a proper license. In addition, the Department of Health and Human Services was directed to prepare a list of agents that would be subject to these regulations. In a Final Rule published in the Federal Register of 24 October 1996 (Vol. 61; #207), these agents were listed and classified as shown in Table 2. The recent anthrax scare and increased concern about bioterrorism since the events of 9/ll have led to increased awareness of the dangers of these agents, to additions to the list of select agents, and to significant new funding for Biosafety Level 3 and 4 laboratories to study these agents.. Those who are authorized to use such agents for legitimate research purposes must now demonstrate, before purchase or transfer of the agents, that they can be destroyed on site.
All of the infectious organisms and agents listed in Table 2 can be digested by alkaline hydrolysis. Protein toxins can be hydrolyzed in the same manner as any other protein. All of the nonprotein toxins, which are sensitive to alkali even at room temperature, would be destroyed when heated to 120ƒC to 150ƒC in 1N - 2N NaOH or KOH. Chemical warfare agents based on nitrogen mustard, as well as most nerve and tear gases, are also destroyed by a hot alkali solution.
Recombinant Organisms and Molecules
Recombinant organisms and molecules that produce or encode for a factor associated with a disease would be destroyed in the same manner as are natural organisms. The same is true of nucleic acid sequences coding for any of the toxins listed in Table 4 and of their toxic subunits.
Applications in Biosafety Level 3 and 4 Laboratories
Digestion of contaminated animal tissues and carcasses, destruction of infectious agent stocks, and sterilization of animal bedding can all be accomplished by alkaline hydrolysis in tissue digestors sized to fit into both single BSL3-4 Laboratories and central waste processing areas within the barrier in large BSL 3-4 faciities. Tissue digestors can be incorporated into the barriers so that contaminated waste can be loaded on the dirty (hot) side and the sterile residue, both hydrolyzate and bone fragments, emptied on the clean side. In addition, because alkaline hydrolysis systems are agitated only by pumping the alkaline solution during the process, all of the ancillary valving, pumping, and control functions in these units can be located outside the barrier, with only pipes and conduits passing through seals in the barrier.
The U.S. Environmental Protection Agency has classified eight commonly used chemotherapeutic (antineoplastic) drugs (Table 3) as ìU-Listed Hazardous Wastesî (40CFR262.22I). Although the U.S. Environmental Protection Agency has not necessarily added to this list any of the new agents that have since been developed, all cytotoxic agents should be treated as hazardous. The alkaline hydrolysis process destroys all of these listed agents, converting them to nontoxic compounds that are completely biodegradable.
Resource Recycling versus Waste Disposal
One truly noteworthy point is that while the animal tissues and carcasses may be called ìwastes,î the sterile hydrolyzate produced from them by alkaline hydrolysis is no longer a waste but a resource. This undiluted hydrolyzate, a 5%-7% solution of amino acids, small peptides, sugars, soaps, and electrolytes, is a valuable and versatile nutrient source that can be used as fertilizer, either liquid or dried and solid, as an additive to composting systems, or as a feedstock for anaerobic digestion biogas generation plants that produce methane, steam, heat, and electric power. Biodiesel applications for the hydrolyzate are also being actively explored.
The State of Illinois has already issued permits for spraying the undiluted hydrolyzate as a fertilizer on growing corn and soybean crops. Indeed, the Illinois Department of Agriculture is now using this hydrolyzate as fertilizer at two of its own facilities where it operates tissue digestors. In Europe, where there are shortages of both sewage treatment capacity and of energy resources, it is most likely that the hydrolyzate will be used for biogas generation. As alkaline hydrolysis becomes the method of choice for destroying the specified risk material (SRM) generated by application of the new USDA regulations for slaughtering cattle, it is likely that large conversion plants for the manufacture of one or more of these secondary products from the hydrolyzate or for the recovery of the energy stored in that solution will become a significant part of the rendering industry.
We have attempted in this article to illustrate the versatility of alkaline hydrolysis as a process for treatment and disposal of a variety of biologic, biohazardous, and hazardous wastes in a manner that is nonpolluting, more efficient and economical than incineration, and capable of producing secondary beneficial resources. We are certain that as we learn even more about this process, its applications will continue to increase in medical and veterinary research, clinical practice, and education, as well as in other industries that produce significant amounts of biologic waste, and that it will become the standard method for treating such wastes rather than being considered an alternative method to combustion of incineration.