Effects of SCI-62® on Bacteria

Introduction

SCI-62® is a special proprietary formulation from Chem-A-Co., Inc. used to control algae and bacteria in various water applications. There are several types of water covered in this discussion, including municipal wastewater, sewage sludge discharge, manufacturing process that produce organic waste material in a lagoon or holding pond, and for fresh water lakes and canals. The active ingredient in SCI-62® is copper, or more accurately, the cupric ion form of copper. Copper appears in nature in one of two ionic forms. An ion is simply a molecule with a minute positive (cation) or negative (anion) electrical charge. Copper has positively charged ions only, Cu+ and Cu++, or a single positive charge or a double positive charge respectively. The cuprous ion has a single positive charge. The cupric ion has a double positive charge.

In nature, most inorganic copper in solution is in the form of complexes. A complex is a combination of two or more inorganic elements. The copper complexes found most frequently in water are carbonate, nitrate, sulfate and chloride, for example copper carbonate, copper sulfate, etc.

These copper complexes are biologically inactive and therefore non-toxic to living organisms. The cupric, or double charged ion, typically will remain uncomplexed in most waters that are not high in pH, alkalinity or hardness, meaning it is in its pure ionic form, Cu++, in solution in the water. This characteristic of the cupric ion is important to the natural ability of copper on exert toxic effects to microorganisms such as bacteria, protozoa and algae that are present under varying environment conditions. It is the cupric ion, and not the cuprous ion, therefore, that must be examined to determine what happens to copper in solution after it has complexed with an organic molecule in the water.

Science is most concerned with the cupric ion because it is “active,” meaning it is in a form where it can react with other molecules. Of greatest interest is the reaction with organic molecules that  collectively form living tissues. The relative ability of a particular organic molecule to grab or “chelate” a cupric ion determines the level of toxicity of copper to that specific organism. There are several factors that increase or decrease the relative toxicity of copper (i.e.., its propensity to be attached to molecules within individual cells) to any particular organism or class of organisms.

However, it is first necessary to understand what the cupric ion does when it comes into contact with an organic molecule and how the cupric ion exerts toxic to bacteria.

The Toxicity of Cupric Ion to Bacteria

Many research studies show that copper has the most common toxicity to microorganisms. When copper salts are dissolved in water, the hydrated cupric ions are formed by the coordination of weak-field monodentate ligands which are primary toxicants. Cupric ions have a high affinity for both oxygen and nitrogen-containing ligands and can also bind to sulfhydryl groups. The presence of these ions at certain sites in the cell will disrupt the normal functions and the integrity of membrane and cellular components (e.g. metalloenzyme and DNA).

There have been numerous reviews of studies on the effects of cupric ions on microorganisms. These have dealt mainly with in vitro studies of the biochemical mechanisms showing how metals exert their effects on microorganisms. The sffects of copper on microorganisms are described as follows:

  1. Growth inhibition to microorganisms including Escherichia coli, S. faeclis, soil bacteria
  2. Morphological changes. Cu++ produced large multinucleate cell with thick cell walls in green algae Arkistrodemus braunni.
  3. Biochemical activities include the following studies:a) Cu inhabited the rate of respiration of a mutant strain of  Bacillus megaterium. b) Cu caused a reduction in the synthesis of RNA and protein in E coli. c) Cu alternated the nitrate reductive pathway, resulting in accumulation of nitrite, increased the phosphorus and decreased the hexosamine content of the cell walls of C. blakesleeane. d) Cu greatly inhibited photosynthesis (but to a lesser than growth) in S. platensis. e) Cu inhibited alkaline phosphatase activity in S. quadricauda.

Cupric ions are attracted to the microbial cell wall and are transferred into the cells. Microorganisms can accumulate metabolic and nonmetabolic metals by precipitating or binding the metals onto cell walls or cell membranes. Microbial walls are anionic owing to the presence of carboxyl, hydroxyl, phosphoryl (chemical components), and other negatively charged sites, therefore, cationic metals rapidly bind to these sites by an energy-independent reaction.

The phosphodiester groups (phosphate compound) of the teichoic acid polymers (carbon and hydro compound) and the carboxyl groups of peptidoglycan (chemical compound) are potent metal coordinators in Gram-positive bacteria. Metal deposition in the walls occurs as a two step process. The initial interaction between the soluble metal species and the reactive chemical group, which is stoichiometric (quantity measure), provides nucleation sites around which there is a secondary deposition of more metal, thereby forming large deposits.

The cell walls of Gram-positive bacteria make up 10-40% of dry weight of the cell, depending on the species and the growth conditions. The cell wall of gram-negative bacteria is chemically and structurally more complex then that of gram-positive bacteria, and the peptidoglycan layer makes up only approximately 2-10% of their dry weight of the cell. An additional layer, termed the outer membrane (OM), is located above the peptidoglycan. The peptidoglycan layer of gram-negitive cell walls also contains sites with which metals can interact. These types of reactive sates in the peptidoglycan are similar in those bacteria. The amounts of metal chelated by Gram-negative cell walls are less than those chelated by Gram-positive cell walls. It can be presumed that this is because the peptidoglycan layer is thinner in Gram-negative bacteria and does not contain teichoic acid, a potent chelator of metals. Although the cell walls of living bacteria have been shown to absorb metals, dead bacterial cells also adsorb metals. There are two binding sites in the membranes. One binding site consists of the phosphoryl groups of the cell membrane. The other site is the carboxyl groups of invertase, a membrane-bond enzymes.

Cupric ions are transferred into the microorganism by the following mechanisms:

  1. Transported via carriers.
  2. By the specific transport of cupric ions complexed with specifically exuded low-molecular- weight ligand.
  3. By the nonspecific transport of cupric ions complexed with substrates that serve as carrier molecules through a transport system specific for these substrates.

Metal ions can also be immobilized in the cell envelope, as the result of the binding of the ions to changed groups in the cell wall. They can also become immobilized, albeit less firmly within capsules and slime layers by absorption.

As we know, metallic elements serve as regulators and catalytic centers for hundreds of cellular reactions. The significance of metals in biological catalysis is underscored by the fact that greater than one-third of all characterized enzymes are metalloenzyme. Thus metals serve important functions in all microorganism metabolism. Cupric ions are involved in the natural selection of aerobic cells and the evolution of metalloproteins and matalloenzyme. This evolution resulted in the development of copper enzymes, such as copper-zinc enzymes, herne enzymes and oxygen-carrying proteins. This is the main reason that aerobic bacteria have more resistance to copper than anaerobic bacteria. For example, aerobic bacteria can fix oxygen by using oxygenase, in which cupric ions are incorporated into the enzymes. In anaerobic environments, denitrifying bacteria reduce nitrite to nitrate reductase. Because Fe and Mo are the components of nitrate reductase, when cupric ions interact with a denitrifying bacteria cell wall, cupric ions will be transferred into the cells and will replace Fe (iron), thus disrupting the function of nitrate reductase and resulting the prevention the formation of ammonia.

Some physicochemical factors That Affsct the Toxicity of Copper to Microorganisms

pH The pH of an environment can affect the toxicity of copper ions to microbes by affecting: a) The physiological state and biochemical activities of microbes and, hence, their reaction to toxic substances. b) The chemical speculation of copper ions, which affects their mobility and ability to bind to cell surface.

Copper forms multiple hydroxylated species as the pH of the solution increase, however, the pH at which these hydroxylated species form varies for each copper. Different hydroxylated forms of copper have different toxicities, and each specific form. affects the adsorption of copper to a charge surface. As the pH of the medium is lowered and the concentration of H+ is increased, H+ can- compete with copper ions for ionogenic sites on the surface of cells. The charge of these ionogenic groups is also influenced by pH, and therefore the affinity of the cell surface for copper ions will also be affected by changes in ambient pH. The special form of organic ligands is also affected by pH, and therefore the complexations of copper ions with organic compounds are generally less toxic than free forms of copper.

Oxidation- Reduction Potential.  The oxidation-reduction potential (Eh) of an environment can affect the availability and toxicity of copper ions. Reducing environments have a negative Eh, whereas oxidizing environments have a positive Eh. Copper ions that are deposited into an environment with a negative Eh may combine with S2- to form insoluble sulfide salts that are unavailable for uptake by microbes and therefore are not toxic.

Water Hardness. Hardness in water is caused by the presence of dissolved alkaline earth ions (e.g. Ca, Mg) together with HCO3- and CO32-. In general, the toxicity of a copper is reduced in hard water.

Clay Minerals. Clay minerals affect the toxicity of copper to microorganisms. Because the charge-compensating cations that are adsorbed on clays can be exchanged by other cations, which include those of heavy metals, present in the environment. In general, clays with a high cation exchange capacity (CEC) are more effective than those with a lower (CEC) in the reducing the toxicity of copper to microbes.

Temperatures. Temperatures affects the toxicity of copper to microbes, presumably as a result of the effect of the temperature on the physiological state of cells, rather than on the chemical special or availability of copper. At temperatures above the optimum for microbial growth, copper toxicity increases.

Copper Ion in SCI-62® SCI-62®  contains a proprietary copper “carrier” that holds copper in solution over very long periods in a wide range of water conditions. The carrier holds both the cuprous and cupric ions in solution. However, the cupric ions, although held in solution, are free to act upon the algae in the water. All other copper based products on the market are chelates, meaning they tie up much of the cupric ions’ through chelate bonds as described previously. SCI-62® delivers 100% of the cupric ions as a free, biologically active Algicide and Bactericide. Every drop of SCI-62® under strict quality control, contains 5% cupric ion within a rigid tolerance of only + or – 0.25%. Therefore, an SCI-62® application provides total control with biologically predictable results.

SCI-62® is a registered Algicide/Bactericide with the United States Environmental Protection Agency (USEPA), and is certified as a drinking water additive by the National Sanitation Foundation (NSF Standard 60)