Share thisAlkaline 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:
Proteins
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.
Lipids
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.
Carbohydrates
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
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).
Indigestible Materials
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.