You Light up My Food Chain: The Process of Photosynthesis

In the last post, the importance of water quality and water chemistry was reviewed in order to determine it’s relevance to the various life forms and life cycles in the pond. In this post, the chemistry that was discussed will be tied into the life processes involved in the pond. This type of science of relating chemistry to biological functions is known as biochemistry. Here, the specific biochemical process that will be elucidated is called photosynthesis.
Photosynthesis is the source of energy for nearly all life forms on earth. It fixes carbon from the atmosphere by taking carbon dioxide (CO2) and utilizes energy from the sun to create organic molecules, such as sugars, which will be utilized in food chains as a source of nutrients. Generally, photosynthesis uses CO2 and water (H2O) to create oxygen (O2) and a sugar (Figure 1). It is used by plants, algae, and cyanobacteria (mentioned in the post Phycology: The Forgotten Field of Study and the Pond). This is the main way that nutrients are created from inorganic molecules to supply food chains. Such an organism with the capability of supplying food chains is referred to as an autotroph. The organisms that cannot create organic materials, and thus get them from ingesting other organisms, are referred to as heterotrophs, which were elaborated in the post Phycology: The Forgotten Field of Study and the Pond. Organisms that photosynthesize are specifically referred to as photoautotrophs, due to their ability to use energy from the sun packaged as photons to synthesize nutrients.

Figure 1: The overall equation of photosynthesis. The reagents are on the left, which are six molecules of carbon dioxide and six molecules of water. Light is utilized to combine these reagents to produce six molecules of oxygen and a sugar molecule, or the products on the right.

Before one gets into the specifics of photosynthesis, one needs to learn a little about chemistry. Redox reactions are a type of chemical reaction that takes place often during photosynthesis and cellular respiration, which will also be explained in greater detail. Redox is a shortened name for reduction/oxidation reactions, which involve the transferring of electrons between atoms and molecules, the building blocks of all the substances in our world. The oxidation portion involves the loss of an electron, and the reduction involves the gain of an electron (Figure 2). It is easy to think of a reduction as a reduction of charge and the gain of an electron in the atom or molecule involved since an electron has a negative charge. In a chemical reaction, everything must balance out; therefore there cannot be an oxidation without its corresponding reduction, or vice versa. In a redox reaction, a reductant, or reducing agent will transfer electrons to an oxidant, and thus an oxidation will occur where the reductant lost an electron and is oxidized. An oxidant or oxidizing agent will gain an electron from a reducer and become reduced in a reduction reaction. This is where the oxidant gained an electron. In other words, an oxidant oxidizes other substances, or removes electrons from other substances, while reducing substances reduce other substances, or give electrons to other substances (Figure 2). Due to this, oxidants are often referred to as electron acceptors while reducing agents are referred to as electron donors. The redox pair in a reaction therefore consists of an oxidizing and reducing agent. A simple example of a redox reaction is the substitution reaction of iron (Fe+2) and copper sulphate (CuSO4) (Figure 3).

Figure 2: The two parts of a redox reaction. The oxidant oxidizes other substances or removes electrons from other substances. Therefore, the oxidant gains an electron and is reduced in the reduction half of the reaction at the top. The reductant reduces other substances. It therefore loses an electron and is oxidized in the oxidation portion of the redox reaction at the bottom.

Figure 3: The substitution reaction of iron and copper sulphate. Copper sulphate (CuSO4) has a copper atom (Cu+2) with a charge of positive two bound to a sulphate ion (SO4-2) with a corresponding negative charge. Copper is reduced or is the oxidant, and therefore accepts two electrons from the iron (Fe) atom. Iron, therefore, is oxidized and is the reducing agent which donates two electrons. Iron initially has a charge of zero, but then loses two electrons to have a charge of positive two and is now bound to the sulphate ion with a charge of negative two.

In this reaction, copper (Cu+2) is bound with sulphate (SO4-2) in one molecule of copper sulphate, but the copper atom in the molecule is substituted for iron and the new compound formed is iron sulphate (FeSO4). The two half equations, which consist of the redox reaction, are:

Figure 4: The redox reactions of the substitution of iron and copper sulphate. Iron (Fe) in the top reaction or the oxidation reaction loses two electrons, denoted by 2e, and is oxidized. Iron is therefore the reducing agent. Copper (Cu) in the bottom corresponding reduction reaction, is reduced and accepts two electrons from the copper atom and is the oxidizing agent.

where the iron is oxidized and the copper is reduced. Therefore, iron is the electron donor and reductant, while copper is the electron acceptor and oxidizing agent.
Photosynthesis involves the transfer of electrons and therefore consists of redox reactions. It begins when light is absorbed by proteins called photosynthetic reactor centres. These centres, in plants, will contain mainly the pigment chlorophyll, but can also contain other secondary pigments such as carotenes and xanthophylls. Algae, which are the other major photosynthesizers of the world, will contain the same pigments in addition to phycoerythrin in red algae, and fucoxanthin in brown algae and diatoms. In the photosynthetic cyanobacteria, the photosynthetic reactor centres are in the plasma membrane, whereas in plants and algae, these centres are located in the chloroplasts. Chloroplasts are organelles, or compartments within a cell, that contain the photosynthetic components and regulate photosynthesis (Figure 5). They are the energy centres of plant or algal cells. A typical plant cell will contain 10-100 chloroplasts, and although all plant cells will have chloroplasts, most of the energy a plant requires will be captured through its leaves.

Figure 5: Plant cells with visible chloroplasts. Chloroplasts are the organelles, or compartments within a cell, that regulate photosynthesis. They often contain the photosynthetic pigment chlorophyll, which is green.

In the chloroplasts, two sets of chemical reactions take place, the light-dependant and light-independent reactions. In the light-dependant reactions, chlorophyll captures energy and facilitates creating molecules with the capability of storing energy. These molecules include adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH), which are molecules with a higher energy state. Later on, when these molecules are converted, energy will be released and can be used for other biochemical processes. The light-independent reactions occur later in photosynthesis. These reactions do not require light like the light-dependant reactions. In these reactions, ATP and NADPH are utilized and the energy they release will assist in reducing CO2. Thus CO2 can be captured and fixed into organic compounds, such as sugars like glucose (Figure 6).

Figure 6: The light-dependant and light-independent reactions of photosynthesis. These two types of reactions are necessary to complete photosynthesis. Light is absorbed by chlorophyll to assist in the formation of the compounds ATP and NADPH, which are molecules high in energy. These energy containing compounds will be utilized in the light-independent reactions of the Calvin cycle. In order to complete the light-dependent reactions, water is used up and oxygen is formed. The Calvin cycle utilizes ATP and NADPH, formed in the light-dependant reactions, to fix carbon in carbon dioxide in order to make sugars.

When light first reaches the chloroplast, the chlorophyll pigment absorbs a unit of energy from the sun, otherwise known as a photon. This causes the chlorophyll to lose one electron and become oxidized and to pass it on to various proteins in the chloroplast thus starting a flow of electrons down an electron transport chain. The ultimate result of the electron transport chain is the reduction of NADP to NADPH, which is a molecule used by photosynthesizers to store energy. The chlorophyll molecule, which has lost an electron to begin the electron transport chain, regains its electron from H20 which plays the role of the reducing agent and donates an electron to the chlorophyll molecule. This releases O2 from the water and creates a proton (H+) gradient across the membrane of the chloroplast which creates a proton motive force. Protons are just another way of referring to a hydrogen atom obtained and separated from water that has lost an electron and now has a positive charge. Therefore, there is a difference in the concentration of positively charged hydrogen atoms across the chloroplast membrane. This gradient of protons is used by an enzyme called ATP synthase to produce the other energy storing compound, ATP (Figure 7). If one thinks about gradients and the natural entropic tendency for gradients to diminish and smooth out, it will become clear how a proton gradient can furnish energy to create ATP. This is seen on a larger scale when a water gradient is stored by a dam. It takes a lot of energy to make a dam and to store the water behind the dam; therefore, potential energy is stored as a water gradient. Consider the force of all the water if it were released from the dam all at once. This energy is used to make hydroelectricity. The same concept applies in chloroplasts to create ATP from a proton gradient (Figure 8).

Figure 7: The light-dependant photosynthetic reactions in the membrane of the chloroplast. On the left, light from the sun causes chlorophyll in PSII to lose an electron and become oxidized. The electron will pass through various proteins in an electron transport chain and will arrive at a point where the molecule NADP can be reduced to NADPH in PSI, which is an excellent chemical method of storing energy for other biochemical processes. Examples of other biochemical processes include the fixing of carbon dioxide in the light-independent reactions of photosynthesis. The chlorophyll in PSII, which has lost an electron, regains another from water (H20) which is oxidized and releases a proton (H+) and oxygen (O2). The proton gradient created is used by ATP synthase, on the right, to create another energy storing compound, ATP.

Figure 8: The proton motive force across a chloroplast membrane. Proton motive force relies on a varying concentration of protons (in blue), or positively charged hydrogen ions across a biological membrane. Like water behind a dam, a higher concentration of these ions on one side of the membrane will possess a potential energy value. When these protons pass through a membrane protein (in red), which plays a role similar to a dam that can be opened and closed, the energy can be used for certain biochemical processes, such as the synthesis of ATP by ATP synthase.

Once the electrons reach the end of the electron transport chain, as the reduced form NADPH, this compound can further provide electrons in order to carry out reductions for other chemical reactions. The reduction reactions that will take place in order to complete photosynthesis include the reduction of CO2 into sugars. The other half of the redox reaction is the oxidation of H20 into O2 as mentioned before (Figures 1, 6 and 7). The reduction of CO2 into sugars consists of the half of photosynthesis that does not rely on sunlight and is therefore light-independent, as described earlier. This part of photosynthesis is referred to as the Calvin cycle. This cycle involves fixing and reducing CO2 in order to form sucrose and starch. These products are then available to be utilized as nutrition by plants, and to produce other organic compounds needed for plant life, such as lipids (fats), cellulose, and amino acids. The Calvin cycle does not require light but it requires electrons, which are donated by NADPH, and it requires CO2 (Figure 6).
Another biochemical process of plants that does not require light is called cellular respiration. This set of chemical reactions, however, is employed by all living cells including animal cells and not just autotrophs. Cellular respiration is a way of utilizing organic nutrient molecules such as sugars to power and sustain cellular life. Cellular respiration is, in many ways, the reverse or opposite of photosynthesis. It is the oxidation of sugars such as glucose, and the reduction of O2 to produce CO2 and water (Figure 9).

Figure 9: The process of cellular respiration. Sugars, such as glucose (C6H12O6), are oxidized and oxygen (O2) is reduced, which are the reagents of cellular respiration. The products formed are carbon dioxide (CO2) and water (H2O). Cellular respiration is the utilization of sugars and O2 by many life forms to fuel countless biochemical processes. Cellular respiration is the reverse of photosynthesis, which takes CO2 and H2O to form O2 and sugars.

Cellular respiration has a similar goal as photosynthesis. It utilizes certain compounds in order to make compounds such as ATP and NADPH, which can transfer energy to make possible an array of biochemical processes.
Photosynthesis is a very important process of all ecosystems including the pond. This is because it will fix carbon, a component of all organic or biological molecules, and it furnishes O2, an important gas necessary for most forms of life because it is utilized for cellular respiration. In a pond, plants will photosynthesize, but an even more important source of O2 and photosynthesis is from the algae in a pond. An excess of algae can be very unsightly, however, some amount of algae is considered important to the pond ecosystem. In fact, an ideal pond will have an overlapping of algal and bacterial species to achieve a proper balance. Algae will fix carbon, from either the alkalinity of the H2O or from a solution of CO2 from the atmosphere, and will furnish carbon compounds and O2 for bacterial replication. The bacteria will then supply certain carbon and nitrogen compounds, which will be of use to the algae (William et al., 1953). Having both algal and bacterial species in a pond will also ensure that there will not be an excess of one or the other population, therefore, population numbers will be controlled. It is important to have a certain concentration of O2 in the pond to sustain life, and this is achieved either through aeration and circulation, or through photosynthesis (Elaborated in the last post “Water, water everywhere! So what are we to think? Water quality and the pond”).
In conclusion, photosynthesis, or utilizing light, CO2 and H2O to synthesize sugars and O2, is a very important process for life in the pond ecosystem among others. Aeration and circulation are methods to ensure proper O2 concentrations of the H2O in a pond, but photosynthesis from plants and algae also play a huge part in this. To ensure a properly balanced pond, some photoautotrophs are necessary. However, if you are contemplating the number of plants or the algal population in your pond, please give us a call at Village Pond and Garden and we will assist you in achieving the right balance of photosynthesizers in your pond.

In the next instalment →Green with Chlorophyll or What Makes Up Plants.
The next instalment shall come in spring. Now it is time for me to take a break and go back to school to study infection prevention and control. Who knows, maybe this might have some crossover knowledge for life in ponds. Either way, learning is fun and I am glad that I could learn and revise these topics with you fellow readers. Thank you!

Summary/Important Points

• Photosynthesis fixes carbon from the atmosphere by taking carbon dioxide and, with water and energy from the sun creates oxygen and sugar.
• Photosynthesis is a source of nutrients for nearly all life forms on earth. It is employed by plants, algae and cyanobacteria.
• Photosynthesis utilizes redox reactions, which involve the transfer of electrons from one compound to another.
• Redox is short for reduction/oxidation reactions. Since chemical reactions must all balance out, a reduction cannot occur without an oxidation or vice versa.
• An oxidation is the loss of an electron while the reduction is a gain of an electron. A reducing agent will transfer an electron to an oxidant and will become oxidized itself. An oxidant will remove an electron from a reducing agent and will become reduced itself. Oxidants are often referred to as electron acceptors, while reducing agents are referred to as electron donors.
• Photosynthesis commences when light is absorbed by the photosynthetic reactor centres, which contain chlorophyll and can contain other pigments depending on the photosynthesizer. The photosynthetic reactor centres are in the plasma membrane in cyanobacteria. In plants and algae, these centres are located in specialized subcellular compartments called chloroplasts. Chloroplasts are the organelles responsible for photosynthetic reactions in a cell.
• Two sets of chemical reactions take place in photosynthesis, the light-dependant and light-independent reactions. In the light-dependent reactions energy from the sun is used to create molecules high in energy. These compounds are called adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). Water is degraded in these reactions and oxygen is released. The light-independent reactions require no light, but utilize ATP and NADPH to fix carbon from carbon dioxide to produce sugars.
• Cellular respiration is the opposite of photosynthesis. It utilizes sugars to power and sustain cellular life. It utilizes sugars and oxygen to produce carbon dioxide and water. All cells utilize this process, not just photosynthesizers.
• Proper oxygen concentrations are essential to maintain a pond’s ecosystem. Circulation and aeration assist in this balance, but photosynthesis also plays a huge part.

References/Further Reading

http://en.wikipedia.org/wiki/File:Photosynthesis_equation.svg
http://en.wikipedia.org/wiki/File:Redox_Halves.png
http://en.wikipedia.org/wiki/File:Plagiomnium_affine_laminazellen.jpeg
http://en.wikipedia.org/wiki/File:Simple_photosynthesis_overview.svg
http://en.wikipedia.org/wiki/File:Thylakoid_membrane.png
http://en.wikipedia.org/wiki/File:Chemiosmosis1.png
Copeland, B.J., Butler, J., L., and Shelton, W., L., 1961. Photosynthetic Productivity in a Small Pond. Proc. of the Acad. of Sci. for 1961, p22-26.
Oswald, W. J., Gotaas, H.B., Ludwig, H.F., and Lynch, V., 1953. Algae Symbiosis in Oxidation Ponds. Sewage and Industrial Wastes, 25(6):692-705.

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Water, water everywhere! So what are we to think? Water quality and the pond.

The last post described the physical methods to control microbial populations, either by bursting microbes, altering their chemical structures within the cells with radiation, by burning them with heat or by slowing their replication by taking heat away (Physical Methods to Kill Microbes or Taking Microbes by Force). Microbes, like the pond enthusiast or any living being for that matter, rely on water for life. This post will go over water quality and how it can affect life in a pond and the overall quality of a pond.
Several criteria are considered for gauging the water quality in a pond or body of water. Here are listed the nine factors of water quality by the United States National Sanitation Foundation. They developed standardized tests and implemented them in 1970 in order to determine water quality.

• Temperature.
• Dissolved oxygen.
• Fecal coliform bacteria numbers.
• Five day biochemical oxygen demand.
• pH.
• Total phosphates.
• Total nitrates.
• Turbidity.
• Total solids.

The first criterion for gauging water quality is the temperature. The temperature is a blanketing characteristic of water and the environment and it will change frequently as the weather does. The temperature of water will affect almost all of the other criteria of water quality. For example, the solubility of certain substances, like oxygen, phosphates, nitrates and organic molecules will increase or decrease depending on the temperature of the water into which these substances are dissolved. This will affect microbial growth and numbers due to the availability of nutrients. Finally, microbial growth itself will be affected by the temperature since higher temperatures will generally increase microbial growth to a point. Increased microbial growth will also change the demand and availability of nutrients in the water. The effect of the temperature will also be elaborated on with regards to each criterion as they are described.
Dissolved oxygen is important to keep at high enough levels in the pond to ensure life of the plants and fish present. Oxygen is required by these life forms, and by many bacteria which utilize oxygen in the nitrification process of one’s pond for example, which will later be described with the nitrogen cycle. Oxygen is crucial to a large number of biological processes. The amount of oxygen that can be present in water will depend on many factors, but one is the temperature of water, as alluded to earlier. Less oxygen is capable of being dissolved in water as the temperature increases. This is why fish kills, due to algal blooms, are more common when the water is warmer. Warmer temperatures increase the growth of algae, thus increasing the oxygen demand, but less oxygen will be available in the first place due to the lower oxygen saturation in warmer water. It is generally considered that the saturation point of oxygen in water is 15ppm (parts per million), or the point where no more oxygen can be dissolved into water. A healthy range of oxygen in a pond will be 5-10ppm, and less than 3-4ppm will result in an unhealthy pond. As mentioned before, fish kills can occur at this point, and other signs of low oxygen in a pond will include unpleasant odours. These odours are created when aerobic bacterial replication and metabolism of nutrients present in the pond becomes reduced, and anaerobic bacteria and their processes increase. These bacteria will create the odoriferous gases of methane and hydrogen sulphide. Oxygen generally enters ponds by way of photosynthesis; a biological process of converting light and carbon dioxide into oxygen and sugars (mentioned in the post “Phycology: The Forgotten Field of Study and the Pond” and this will be elaborated in the next blog entry), wind, waves, and aeration.
The third criterion is the number of coliform bacteria, which are less important for water quality in the pond, but play a huge role in drinking water quality. Coliform bacteria are a type of bacteria found in large numbers in the feces of warm blooded animals and assist in digestion, which is why they are usually referred to as fecal coliform bacteria. They are rod shaped in appearance and are incapable of forming the protective spores that some bacteria can employ, which was described in the last post (Physical Methods to Kill Microbes or Taking Microbes by Force). Coliform bacteria include such microbes as Escherichia coli, but the larger problem with coliform bacteria is that some types can be a lot more harmful than others. One example is the strain of E. coli, O157:H7, which caused the outbreak in Walkerton, Ontario, where at least seven people died and about 2500 became infected in the year 2000. These bacteria, when ingested, can cause illnesses such as diarrhoea and even life threatening bloody diarrhoea in extreme cases (also known as enterohemorrhagic diarrhoea). It goes without saying that zero numbers of these microbes are ideal for drinking water.
Although there is little tolerance for microbes in drinking water, microbes will be present in all other types of water, including pond water. The numbers of microbes present will affect the biochemical oxygen demand (BOD). The BOD is the amount of dissolved oxygen necessary for aerobic microbes to break down the organic substances in a body of water at a certain temperature over a certain period of time. The time period of five days was selected due to the fact that a large enough amount of oxygen was used in this period of time to convert organic matter in the pond without affecting the reproducibility of the results. Generally, 68% of the BOD is used in five days at 20°C and larger amounts of the BOD will be converted in 10 (90%) and 20 days (99%). However, the microbial population will shift to nitrifying bacteria, which will affect the results of the test. Nitrifying bacteria are the bacteria that use oxygen to convert toxic ammonia compounds to nitrates in the nitrogen cycle, which will be described in greater detail. Since BOD measures the amount of oxygen used up in an amount of time by microbes in the water, it is a gauge for the degree of organic pollution present. This is because of the fact that the more organic compounds that are present for microbial reproduction, the more demand there will be on oxygen, and the BOD will increase. BOD will not be able to be measured accurately if the water may contain products that may hinder microbial reproduction, such as antibiotics, sanitizers, chlorination, or odour control formulations, which can be found in certain waste waters, depending on the source. BOD is generally not used for determining the quality of drinking water, but rather for waste water for example, from waste water treatment plants or from rivers, lakes and streams. Pristine rivers will have a BOD of less than 1mg of oxygen consumed per litre of sample (1mg/l) during five days, at 20°C. Treated sewage will have a BOD below or equal to 20mg/l, and untreated sewage, generally has a BOD of in the range of hundreds of mg/l of water.
A ubiquitously important quality of water, whether it is inside organisms involved in their biological processes or in the environment, is pH. The pH is a measure of the relative acidity of water. Water, and compounds that dissolve in it, have a natural tendency to ionize, which means that a small fraction of water will dissociate and form ions:

Figure 1: The dissociation of water (H20) into a positively charged hydronium ion (H3O+) and a negatively charged hydroxide ion (OH-).

Compounds that dissolve in water will also dissociate and ionize. The ion concentrations will affect the relative acidity of the water. Hydronium (H3O+), in larger numbers, will make the water more acidic. The pH of the water will change depending on what is dissolved in the water, be it an acid, such as sulphuric acid (H2SO4):

Figure 2: The dissociation of sulphuric acid (H2SO4) into water. Ionized products on the right (HSO4 and H3O+) will form when dissolved in water.

Or a base, such as ammonia (NH3), which can accept hydrogen from a hydronium ion found in the solution:

Figure 3: The dissociation of ammonia (NH3) into water. Ionized products on the right (NH4+ and OH-) will form when dissolved in water.

For example, water that is exposed to air is generally mildly acidic because it absorbs the carbon dioxide (CO2) in the air, which is slowly converted to carbonic acid (H2CO3), and will further be converted into the ionized products (HCO3- and H+) thus making the solution more acidic.

Figure 4: Carbon dioxide dissolving in water. First, carbonic acid is formed (H2CO3), then the ionized products are dissolved in the solution (HCO3- and H+).

Pure water with no acidity or alkalinity is considered to be neutral at a pH of 7 at 25°C. The pH is charted on a logarithmic scale, which is exponential, so a difference of one pH unit is equal to a tenfold difference in the hydronium ion concentration. The scale goes from 0-14, where values below 7 are acidic and those above 7 are basic or alkaline. Alkalinity, however, is not always interchangeable with the term basic. Whereas the pH will always measure the quantitative number of hydronium ions dissolved in water, alkalinity is the capacity of a solution to neutralize an acid or the amount of acid that can be added to a solution before the pH changes drastically. A solution can be alkaline but it does not have to be strongly basic (have a high pH), even though the terms alkalinity and basic are often interchanged. Some factors that can influence the alkalinity of a solution are carbonate, bicarbonate, hydroxide, phosphate, silicate, nitrate, ammonia and sulphide.
Another chemical factor that is important in water quality is the amounts of phosphates and nitrates present, especially where plant and algal growth is concerned. They can speed the growth of algae in the pond and thus disrupt the natural balance of a pond. Waterfowl excrement contains a lot of both compounds, and having even one or two birds contributing to their waste to a pond can disrupt the pond’s ecosystem in certain conditions. The factors that can greatly assist or hinder in this include the size of the pond, the temperature, the circulation, and what microbes are present and whether or not they are part of a bioaugmentation program (as discussed in “You Get More Flies with Honey and You Get Better Microbes with Probiotics”). Phosphates are a large contributor to plant growth. Therefore, they can be found in many plant fertilizers and will also contribute to algal blooms. The water quality is thought to decline when the level of phosphates reaches 0.05-0.1ppm. Phosphates, as well as in waterfowl excrement and in fertilizers, can also be found in pesticides, dead vegetation or landscape debris, and water runoff after rain. Once phosphates are present in a pond, they are quite difficult to remove. One method used to remove phosphates is barley straw. When bacteria break down barley straw, hydrogen peroxide is produced. Hydrogen peroxide is an algaecide and an oxidizing agent that will bind phosphates and remove them from the water. Phosphates can also be removed by the addition of alum (aluminum sulphate or potassium aluminum sulphate). Once the alum binds to the phosphates and forms aluminum phosphate, it will precipitate the phosphates out of the water since aluminum phosphate has a lower solubility than aluminum sulphate. Alum is a widely used method to control phosphate levels in ponds; however, in shallower ponds it is less ideal since it can leave these ponds with a milky appearance.
Nitrate and phosphate levels often go hand in hand, having similar origins in excrement, decomposing plant and animal matter, and water runoff. Though phosphates, as mentioned earlier, are extremely important when contributing to algal growth, nitrogen levels in a pond will also contribute. Nitrogen, like phosphates and oxygen, is extremely important for life, therefore, some nitrogen is necessary for a balanced pond ecosystem, but there can be a point where the ideal nitrogen levels are exceeded, which is above 5-7ppm. Nitrogen is present in large quantities in the atmosphere, 78% of it consists of nitrogen. Though there is a lot of nitrogen available in the atmosphere, there are limited ways for biological creatures to harvest it. The manner in which nitrogen enters the food chain is by nitrogen fixation. Some bacteria, such as Rhizobium species, which live on the root nodules of legumes, and some free living bacteria, such as Azobacter species, have the capability to take nitrogen from the air and incorporate it into biological molecules. In fact, Rhizobium lives in a symbiotic relationship with some legumes contributing nitrogen in the form of ammonia to the plant in exchange for carbohydrates. These bacteria possess an enzyme called nitrogenase that will combine gaseous nitrogen (N2) and hydrogen (H) to produce ammonium (NH4+). This is the start of the nitrogen cycle (Figure 5). It is referred to as fixation, where bacteria take nitrogen from the atmosphere and convert it to compounds such as ammonia, nitrite (NO2-) and nitrates (NO3-). Then the compounds made by the bacteria are absorbed and used by plant life, this is called assimilation. As plants and animals then decompose, ammonium is formed once again. This is the ammonification portion of the cycle. Ammonia then can re enter the food chain being absorbed by plants, or digested by bacteria such as Pseudomonas and Clostridium species. These species, when oxygen is absent, utilize nitrogen instead of oxygen for certain biological processes and will produce nitrogen gas in the step called denitrification. Thus, the nitrogen cycle completes itself by either re contributing the nutrients to plants and bacteria at the base of the food chain, or by reconverting nitrogen to its original gaseous atmospheric form (Figure 5).

Figure 5: The nitrogen cycle. Atmospheric nitrogen (N2) is brought into the ground and the nitrogen cycle by way of bacteria either living symbiotically on the roots of certain legumes, or as free living bacteria in the soil. This step is referred to as nitrogen fixation, which results in the nitrogen being incorporated into ammonium (NH4+). Ammonium is further converted to nitrites (NO2-) and, finally to nitrates (NO3-) by other bacterial species. These nitrates can be absorbed by plants in the process called assimilation, since the nitrogen is in a form that is capable of being utilized by plants. Then, nitrogen can be utilized by herbivores and carnivores. As the plants and animals produce waste, or die and decompose, they produce ammonium to contribute to the ammonification of the nitrogen cycle. Finally, certain bacterial species, in the absence of oxygen, can take certain nitrogen containing compounds, like nitrate, and convert it to gaseous nitrogen. Denitrification therefore completes the nitrogen cycle.

The nitrogen cycle exists in many ecosystems including the pond (Figure 6). Nitrogen enters the pond by the bacterial fixation of nitrogen and will be used and excreted by plants and fish. Then ammonia is produced, which is toxic to these life forms. Then, certain bacteria present will be able to convert ammonia to nitrite, which is equally toxic, and finally to nitrate, a far less toxic substance. Nitrate can then be denitrified in a pond by the denitrifying bacteria. This process though, as mentioned earlier, is an anaerobic process meaning it has to happen in the absence of oxygen. Therefore, in order to keep a healthy pond, a bottom layer of sediment is recommended to facilitate denitrification. Oxygen, as mentioned earlier, is also required for healthy levels of certain nitrogenous compounds and for the continuation of the nitrogen cycle. Without sufficient oxygen, nitrification cannot be completed and the toxic compound, nitrite, will accumulate in a pond.

Figure 6: The nitrogen cycle in a pond. Fixation of gaseous nitrogen and conversion from ammonia to nitrites and nitrates is done by bacterial species. Among them are the Nitrosomonas and Nitrospira species. Nitrates are then utilized by plants for nutrients, which will contribute to the food chain, be it for fish or other organisms in the pond. Once plants and fish die, or produce waste, ammonia is produced which will continue the nitrogen cycle in an aquatic environment.

The final criteria for water quality are turbidity and total solids. Turbidity is the cloudiness of water caused by particulate matter suspended in it. Its effect is similar to that of smoke in the air. Some matter will be large and heavy enough to eventually settle on the bottom, but until then will contribute to the turbidity. Very small solids will settle much more slowly and sometimes not at all if the sample is agitated enough and will remain contributing to the turbidity of the water. The turbidity can be increased by phytoplankton, soil erosion in water runoff and precipitation of certain compounds such as phosphates. The turbidity, as it increases, will decrease the amount of light that can enter the depths of water. This may decrease the levels of photosynthesis from plant life, thus decreasing the oxygen levels. Turbid water also becomes warmer more readily since suspended particles will absorb heat from the sun, which in turn affects many other aspects of water quality. High turbidity can also hinder the ability of fish gills to absorb oxygen. Though high turbidity is often considered bad, some turbidity is beneficial as it indicates some microbial life in the pond and thus a balanced, functional ecosystem. High turbidity, in some cases, serves as protection for certain life forms like juvenile fish in mangroves (Blaber, S.J.M., 2000).
The total dissolved solids are similar to turbidity in that it involves particles being suspended in the water. However, the particles are much smaller, the standard being that the particles should be able to pass through a 2μm filter. The total dissolved solids involve all inorganic and organic substances dissolved in the water, including molecular, ionized and micro-granular suspended forms. Though the source of the total dissolved solids is from water runoff and soil erosion, the same as that of the impurities that increase the turbidity of water, these dissolved solids are generally not associated with health risks and are considered more for aesthetic purposes when considering drinking water. This measure of water quality is exclusively used for fresh water, since salt water includes the ions constituted as total dissolved solids. Some common constituents include calcium, phosphates, nitrates, sodium, potassium and chloride. The United States National Sanitation Foundation standard for drinking water is less than, or equal to, 500mg/l of water.
Once the ecosystem of the pond becomes unbalanced in one form or another, there are methods that one may employ to try and bring back the balance. It is always recommended though to employ these methods as preventative measures to balance an already healthy ecosystem. This is because treatments, though they may suppress surplus growth, like an algal bloom, will only temporarily do so and such imbalances will occur in a cyclical fashion. For example, if a copper compound is added to control algal growth, dead algae will then accumulate and decompose. This will free up nutrients for the next algal bloom. During this process, residual chemicals from the treatment will also accumulate. Therefore, though there are compounds available to control surplus growth of algae, such as copper carbonate and chelated forms of copper, these generally offer only a temporary solution. In the long term, maintaining a balanced ecosystem will be much more beneficial. Chemical treatments may also differ for each pond because of the various aspects of water quality, such as the pH, temperature, and the levels of organic materials will affect the manner in which these treatments function.
A good way to maintain a pond ecosystem is by aeration. Aeration is crucial for ensuring maximal levels of oxygen in the pond, but is also useful for distributing the water chemistry, such as the temperature, pH, nitrates, phosphates, and microbes. There exist a few tools to assist in the aeration of a pond. These include floating fountains, a water pump with an intake at the bottom of the pond, and compressors. Floating fountains can increase circulation and provide aeration, but are designed more for aesthetics and only affect a small area of the pond near the surface whereas, ideally, one would want the whole pond evenly affected by aeration and circulation. Water pumps with an intake at the bottom of the pond affect a larger area, removing and circulating water at the bottom of the pond and discharging it in a fountain or waterfall, however, it will only circulate the water in a channel, leaving dead areas outside of the water flow. Compressors, unlike the other two methods, are designed purely with aeration in mind and not so much aesthetics. These compressors are installed at the surface of the pond with air lines extended to the bottom. The bubbles of air that form then float to the surface and as they do, they transfer oxygen and circulate water throughout all depths of the pond, creating a continuous circulation pattern. These will not affect the appearance of the pond either, since the system is designed to make very small bubbles because smaller bubbles will circulate more water and will transfer more oxygen than larger bubbles. Some methods may be better than others, depending on the pond and the preferences of the pond enthusiast, but combinations of these methods are generally preferred.
Another excellent method of maintaining a pond ecosystem is by bioremediation. This was the topic of the post “You Get More Flies with Honey and You Get Better Microbes with Probiotics”. It includes adding specific microbes in order to obtain balanced pond water chemistry, and to assist in maintaining a proper ecosystem, such as by preventing algal blooms. Sometimes, even with the best of the pond enthusiast’s efforts, a pond can become improperly balanced in its water chemistry. If this is the case, let us at Village Pond and Garden look at the various factors that may contribute to this. Even if it is a healthy pond, we can discuss ways to keep it this way. For we at Village Pond and Garden also believe that prevention is the best method to having a healthy pond ecosystem.

In the next installment →You Light up My Food Chain: The Process of Photosynthesis

Summary/Important Points

• The criteria for gauging water quality set by the United States Sanitation Foundation are the temperature, dissolved oxygen, fecal coliform bacteria numbers, five day biochemical oxygen demand, pH, total phosphates and nitrates, turbidity and total solids.
• The temperature can affect the solubility of compounds that will affect the balance as well as the growth rate of organisms in the pond ecosystem.
• The amount of dissolved oxygen is important to maintain, for it is necessary for many biological processes in the pond.
• The number of coliform bacteria is suited to gauging the water quality of drinking water, since some types of these bacteria can cause quite severe illnesses.
• The five day biochemical oxygen demand is the amount of dissolved oxygen necessary for the microbial population to break down the organic substances in a body of water. It reflects the amount of organic pollution in a body of water.
• The pH is ubiquitously important in many chemical and biological processes. It is a quantitative measurement of the relative acidity of a solution, which is affected by the number of hydronium (H3O+) ions present. Pure water has a pH of 7, acidic solutions have a pH below 7, and basic solutions have a pH above 7. The pH is measured on a logarithmic, exponential scale of 0-14.
• Nitrates and phosphates are necessary to all organisms, but it is important to maintain the levels of these compounds in order to have a balanced pond ecosystem.
• Turbidity is the cloudiness of water caused by particulate matter suspended in it. Turbidity can be increased by phytoplankton, soil erosion and the precipitation of certain compounds.
• Total solids are much smaller particles (≤2μm) suspended in the water and are generally for aesthetic purposes. This is exclusively a criterion for fresh water, since some total solids are actually the regular constituents of salt water.
• Certain treatments exist to aid in controlling imbalances in the pond ecosystem, but such solutions are only temporary, delaying a much larger problem that will eventually occur. Preventative methods are recommended such as bioremediation, circulation and aeration methods.

References/Further Reading

http://www.omegalakeservices.com/The_Pond_Ecosystem.html
http://en.wikipedia.org/wiki/File:Aquarium_Nitrogen_Cycle.svg
http://en.wikipedia.org/wiki/File:Nitrogen_Cycle.svg
Blaber, S.J.M, 2000. Tropical estuarine fishes: ecology, exploitation and conservation. Oxford: Blackwell Science.

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Physical Methods to Kill Microbes or Taking Microbes by Force

In previous posts, the control of a microbial population such as in a pond was discussed by using chemical means or disinfectants (“How to Properly Use a Cleaner – Things One Should Know About Disinfectants”), and by using biocontrol or by adding viable microbes as with probiotics (“You Get More Flies with Honey and You Get Better Microbes with Probiotics”). This post will go over another means of controlling microbes, such as the physical means used to kill microbes, or utilizing forces and energy to control their populations. Many of the physical means to kill microbes are not used in the case of the pond enthusiast, but some are, and those that are not still have valuable applications in science, medicine, and in food processing. So, a brief description will be given for each one and its applications. Finally, how these apply to the pond enthusiast will be discussed.
The most common physical method used to treat microbes is by heat. Heat kills microbes by drying them out and by denaturing the proteins within the microbial cells. Protein denaturation is the process where proteins lose their usual three dimensional shapes. In order for a protein to function biologically, it must retain its specific shape (Figure 1). Therefore, heat inactivates proteins within a cell. Several methods utilizing heat are used:

Figure 1: Protein denaturation. The protein on the right maintains it’s biologically active, three dimensional shape with various chemical interactions. Certain conditions, like excessive heat, will cause the protein to lose its form, as seen on the left, and become denatured and biologically inactive.

1. Incineration (>500°C):
This is simply killing microbes by burning them. It vaporizes organic matter but destroys many other substances other than microbes in the process. This is utilized in the lab for sterilizing inoculating wires used to inoculate new microbial cultures in order to ensure there is no contamination in microbiological and other types of experiments.
2. Boiling (100°C):
Doing this for 30 minutes will normally kill all microbes except for some endospores. Endospores are a stripped down, dormant form that some bacteria can adopt when conditions are unfavourable. Bacteria as endospores can remain dormant for a long time until favourable conditions trigger a bacterium to exit the endospore state and enter the active or replicative vegetative state. Endospores pose a large challenge in sterilization because they are highly resistant to many adverse effects like heat and pressure. In order to completely sterilize and kill the endospores, boiling for very long periods of time (>6hrs) is recommended or for intermittent periods of time with cooling.
3. Autoclaving (121°C/15psi/15 minutes):
This is a very efficient method of sterilization. It is used very commonly to sterilize biohazardous waste, surgical dressings, microbiological media, and other liquids in hospitals and laboratories. This method employs heat with pressure in a synergistic fashion to kill microbes. The steam created in an autoclave can reach higher than normal temperatures due to the high pressure. Due to this, autoclaving is very effective at killing the heat and pressure resistant endospores. The standard autoclave cycle is 121°C at 15psi (pound per square inch) for 15 minutes. Therefore, it is a very fast method to completely sterilize objects. The principle of autoclaving comes from the concept of latent heat of vaporization. This means that steam contains much more energy than boiling water. For example, it takes about 80 calories of energy to boil 1 litre of water but much more energy to convert the same amount of boiling water to steam, 540 calories. Therefore, steam contains much more energy and heat to kill microbes.
4. Dry heat or a hot air oven (160°C/2hours or 170°C/1hour):
This is what is used when cooking food, essentially. It does not utilize the more effective wet heat of an autoclave, so longer treatment times are needed. This method is employed to prepare food, and to sterilize glassware, and metal objects. Essentially, objects that will not melt at these high temperatures can be sterilized in a hot air oven.
Cold can be considered a physical method of controlling microbes though it is not a specific force but rather a lack of one, heat. The cold, conversely, does not sterilize or kill microbes but simply slows down or ceases microbial growth. Microbes that have been cooled will generally resume replication upon returning to more ideal temperatures. This method is very popular for food preservation as most of us who have a refrigerator or freezer will know. Many bacteria that do not require oxygen for survival, or otherwise known as the strict anaerobes (ex: Clostridium perfringens and some strains of Clostridium botulinum, or the bacterium that causes botulism), cannot reproduce below 12°C. However, some types of bacteria (ex: Listeria monocytogenes and Yersinia enterocolitica) can replicate below 1°C. Some microbes which are commonly associated with food spoilage can still multiply, albeit slowly, at temperatures as cold as -7°C. At -18°C, however, food can be properly stored since all microbial growth ceases at and below this temperature. Slow loss of quality to food will still occur, but this is not due to microbes but rather from enzymatic degradation in the food.
Other forces used to kill microbes include pressure. High pressure is a well established, non-thermal method used to kill bacteria, yeasts and moulds. Aside from bursting the cells, high pressure alters macromolecules (ex: proteins, polysaccharides), which are present in microbes among other organisms, but it has little effect on smaller molecules. Since the chemical compounds which affect flavours and odours are often smaller molecules, pressure is an excellent force used to pasteurize foods like fruit juices, jams, and dressings. Pressure is also used in the laboratory to kill and burst microbes to study their insides. The French Press is the machine used to do this in the laboratory. It forces a pressurized cell suspension through a narrow orifice (Figure 2). The pressure drop, once the suspension reaches the orifice, combined with the shearing forces, causes the necessary damage to open up the microbes.

Figure 2: The French Press. Pressure is applied from the top and the cells are forced through a narrow orifice at the bottom. The pressure drop once the cells reach the orifice, and the shearing forces, causes the cells to burst open.

Sonication is another method used in the laboratory to burst open cells. This method employs sonic energy. The cells, which will be burst open, are suspended in water and sonic pressure waves are applied, which causes the rapid formation and collapse of micro bubbles. These bubbles, when they collapse, generate shock waves that will disrupt the cell walls of the microbes.
Aside from pressure, heat and sonic energy, yet another force can kill microbes. This force is osmotic pressure. Osmotic pressure often deals with biological processes because it involves a semi-permeable membrane like the cell membranes that living things possess. Small molecules like water can traverse this membrane so, when a concentration gradient like that caused by the presence of a salt or sugar exists across this membrane, the water will pass through this membrane in order to equalize the concentration gradient. Therefore, taking a cell from a solution higher in salts or sugars, which will allow the chemicals to slowly permeate into the cell since they are small enough molecules, then transferring the cells to distilled water will result in a rapid influx of water into the cells due to an osmotic gradient, and the cells will burst (Figure 3). This is often used to break open mammalian cells in the laboratory. Bacterial and fungal cell walls are stronger and will generally not burst due to osmotic pressure. Osmotic pressure is used to preserve food by adding sugars or salts to foods. Though microbes may not burst and die from this, adding sugars or salts will result in essential water being removed from the microbes, since there will be a higher concentration in the solution and thus a concentration gradient.

Figure 3: The effects of osmotic pressure on red blood cells. In the centre panel, the osmotic pressure is perfectly balanced and the water entering and leaving the cells is equal. On the left, water leaves the cells and they dry out, due to a higher concentration of solutes outside of the cell. On the right, water enters the cells and they burst due to a higher concentration of solutes inside the cell.

Finally, come some physical methods used to control microbial growth in the pond, one being filtration. Filtration is simply passing a liquid or gas through cavities where, depending on the size of the cavities, certain impurities or contaminants cannot pass through. Filters are used to remove unwanted substances in our air when we are in certain buildings (office buildings, laboratories, hospitals, etc.). Filters in a laboratory setting can be used to sterilize solutions that would otherwise be destroyed if heat were used to sterilize them (ex: enzymes and vaccines). However, these filters are extremely fine in order to catch bacteria that are usually one micron, or 0.001 millimetres in size! Filters are also used in ponds, not necessarily to eliminate microbes, but to remove larger particulate matter that may detriment the appearance of a pond. Filters, in this case, actually serve as a haven for the biocontrol microbes that may be added to a pond to regulate microbial growth (As reviewed in the last post “You Get More Flies with Honey and You Get Better Microbes with Probiotics”). The biocontrol microbes will get caught in the filter and will colonize it, which is necessary for the microbes to achieve high enough numbers.
Another physical force used in ponds to control microbial populations, which is also used in the food processing industry and in medicine, is irradiation. What one needs to know about light, otherwise known as electromagnetic radiation, is that it exists at many wavelengths. Only a small fraction of light, or wavelengths, are visible. There are other forms of invisible light; this includes X-rays, gamma rays and ultra violet (UV) light. These forms of light have very short wavelengths and possess a lot of energy, and due to this, can be damaging to life forms (Figure 4).

Figure 4: The electromagnetic spectrum. The wavelengths of the electromagnetic radiation are represented by the wave on the top and the approximate size of the various waves is scaled to various objects on the bottom. Visible light makes up only a small fraction of electromagnetic radiation in the centre. The higher energy wavelengths, such as UV, gamma, and X-rays, are shorter and are situated on the right.

They exert their effect primarily by ionizing water molecules to form highly reactive hydroxyl radicals, which are compounds that can react and change the structures and compositions of other molecules present, some of which may be essential to normal biological function. UV light also causes damage by altering the structure of DNA, the genetic code of most organisms and the molecule essential for the replication of these organisms. UV light causes units in the code, called thymines, to dimerize or simply to bind together in an abnormal fashion (Figure 5). Once the thymines are dimerized, enzymes that would normally replicate the DNA when the cell replicates are unable to do so, thus cell replication is hindered. UV light is used in some pond filters. As water is passed through the filter, planktonic or free floating algae and bacteria come into contact with the UV light and are killed, should the conditions be ideal. However, algae and bacteria found in biofilms (as described in the second post “No Microbe is an Island: Biofilms”) are unaffected by the UV light since they grow on a surface in the pond and will not have a chance to pass through the filter.

Figure 5: Thymine dimerization. UV light, shown coming in on the left can dimerize thymines, which comprise part of the genetic code of DNA. On the left the thymines, shown in yellow, are bound to their normal counterparts in green. UV light can assist in binding these thymines together to form a dimer, seen on the right, which will alter the form of DNA and inhibit replication of the molecule when the cell divides.

In conclusion, there are many forces that can be used to sterilize or control microbial growth. Some, such as radiation and filtration, in addition to being used in the food processing industry, in laboratories, and in medicine, are also used to treat ponds. Ponds may have filters equipped with UV lights to aide in controlling microbial growth, however, not all ponds have filters, and not all filters have UV lights. Even if a pond is equipped with both, only regular maintenance will ensure the maximum efficiency of both. Filters may become plugged and dirty, and the UV light bulbs may also become dirty, reducing their efficaciousness, or burn out. If you, the pond enthusiast, is not certain whether or not your filter system with UV light is functioning best, or if you may want such a system installed, call us at Village Pond and Garden and we can discuss filter options with you.

In the next installment → Water, water everywhere! So what are we to think? Water quality and the pond.

Summary/Important Points

• The most common physical method used to kill microbes or control their replication is by heat (Ex: Incineration, boiling, autoclaving, and dry heat).
• Heat functions by drying out microbes and by denaturing their proteins. Denaturing proteins is when the configuration, or the three dimensional shape of the protein is lost. This shape is necessary to retain the biological activity of the protein.
• Cold is a physical method of controlling growth as seen commonly with refrigerators and freezers, but it does not kill microbes. It only slows or temporarily ceases their replication.
• Pressure and sonication, or sonic energy, are also used to kill microbes and burst them open.
• Osmotic pressure is caused by a concentration gradient across a semi-permeable membrane, as seen with cell membranes in living things. While the concentration gradient is being equalized, it can result in the cells bursting and dying due to an influx of water, or it can remove essential water and detriment the cells.
• Filtration and irradiation are used in ponds and in other applications, in some cases to control microbial populations. Filtration does not damage microbes but simply removes them from the solution, whereas the high energy electromagnetic radiation, such as UV light, actually does damage to the components of a cell.

References/Further Reading

Gould, GW. 2000. Preservation: past, present and future. British Medical Bulletin. 1:84-96.
Grabski, AC. 2009. Advances in Preparation of Biological Extracts for Protein Purification. Methods in Enzymolgy. 463:285-303.

Protein denaturation http://en.wikipedia.org/wiki/File:Protein_folding_schematic.png
French Press http://en.wikipedia.org/wiki/File:French_press.gif
Osmotic pressure http://en.wikipedia.org/wiki/File:Osmotic_pressure_on_blood_cells_diagram.svg
Electro-magnetic spectrum http://en.wikipedia.org/wiki/File:EM_Spectrum_Properties_edit.svg
Thymine dimerization http://upload.wikimedia.org/wikipedia/commons/f/fd/DNA_UV_mutation.svg

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You Get More Flies with Honey and You Get Better Microbes with Probiotics

The previous posts of this blog have gone over what microbes are, how one will find microbes in their pond, and finally a few methods in which to eliminate microbes. Though it is impossible to completely eliminate microbes from a pond or any environment, the numbers can be reduced by the use of cleaning agents (How to Properly Use a Cleaner – Things One Should Know about Disinfectants). There is also another way in which microbes can be controlled if not necessarily eliminated. This may be with the use of probiotics.
Lately, there has been a lot of buzz about probiotics. Many attribute health benefits to their ingestion, such as those that may be found in some buttermilk, sauerkrauts, fermented cereals, salamis and yoghurts. Probiotics have been found to reduce the incidence and duration of rotavirus-caused diarrhoea in infants and diarrhoea that is caused by taking antibiotics in adults. The effects of probiotics and how this is caused are recently coming to light in science, but what can the pond enthusiast gather from this? How can a pond be cleaned and maintained by adding more germs?
As always, first comes defining what a probiotic is and the terms associated with the subject. The term “probiotic” was first used in 1965 to describe “substances secreted by one microorganism which stimulates the growth of another”. This definition was used mainly to contrast antibiotics which, instead of stimulating, inhibit the growth of another microbe. A more modern definition is that a probiotic is a viable mono- or mixed culture of microorganisms which applied to animals or humans, beneficially affects the host by improving the balance of the bacteria found in the gut. Some though deem that probiotics may not necessarily be viable, but can be simply any microbial preparation that would benefit the host upon ingestion. Generally, though, probiotics are comprised of live microbes and therefore the constituents of microbes such as polysaccharides, proteins and DNA, and dead microbes are often excluded from the term “probiotic”. Another term may encompass the non living components of probiotics, but only if they fulfill the requirement of being able to select for the growth of specific microbes in the gut of a host. This is a prebiotic. A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth of one or a limited number of bacteria in the colon to improve the health of the host. Many prebiotics can come from bacterial preparations, but some come from plant, and yeast preparations. The prebiotic is often mixed with the probiotic in a single preparation so the microbe which the prebiotic is intended to stimulate is already present for maximal results. This is referred to as a symbiotic, which alludes to the synergism one hopes to obtain by adding both products simultaneously.
Prebiotics and probiotics, therefore, are intended to increase a certain microbe in numbers in order to elicit a beneficial effect. Not everything is yet known on how the increased presence of probiotics can elicit this effect, but some ideas are in place. The possible modes of action include:
1. The production of inhibitory compounds:
Some microbes produce antibiotics, hydrogen peroxide, siderophores (compounds that bind to iron reducing its availability for other microbes which require it), organic acids, ammonia, diacetyl, enzymes which can interfere with the functioning of other microbes and degrade them (proteases, lyzozymes), bacteriocins (protein toxins produced by bacteria that inhibit the growth of similar or closely related bacterial strains), and can alter the pH of the environment. All of these products of probiotics can make an environment unfavourable to other microbes while ensuring the probiont has a replicative advantage. The idea of microbes competing in the same environment was already discussed in the second post “No Microbe is an Island: Biofilms”, so the idea of microbes having a competitive advantage in a mixed culture over others is no new concept. Probiotics simply would utilize these bacterial assets to the benefit of the host or pond enthusiast.
2. Competition for chemicals, available energy and adhesion sites:
Adding probiotics to increase their numbers may limit nutrients and energy sources for bacteria that may later cause a problem such as pathogenic bacteria in the gut. In the instance of gut bacteria, they may also take up the limited adhesion sites needed to permit the colonization of a host.
3. Alteration of microbial metabolism:
The introduction of probiotics to an environment has been shown to alter the way bacteria already present utilize nutrients, but how this produces favourable outcomes has yet to be determined.
4. Stimulation of host immunity:
The addition of probiotics may result in increased resistance of the host to pathogens. Some believe that this may be because the presence of probiotics can prime a host’s immune system for when the actual pathogen will present itself, though further research must be done to elucidate these mechanisms.
5. Enzymatic contribution to digestion:
A way that probiotics may be beneficial is that their enzymes, which may not be present in the host, can digest certain compounds that would otherwise be indigestible. This would therefore provide a new source of nutrients. Enzymatic digestion from probiotics may also contribute to the breakdown of toxic compounds.
6. Source of macro- and micronutrients:
Enzymatic digestion may assist in creating a source of nutrients, as mentioned in the previous reason, which can be a source of micronutrients for the host and the pond. Macronutrients, such as vitamins, may also be synthesized by probiotics which may produce positive results.
Most of the examples given so far have been with respect to probiotics being ingested to benefit a host like humans, but how do probiotics and their possible uses apply to a pond? In aquaculture, or in a pond, the microbial habitat undergoes many more alterations than in the more sheltered habitats within the host gut. In a pond, depending on the time of year, weather and life forms that may already be present, factors can fluctuate like the salinity, temperature and oxygen concentration. Also, microbes present in ponds will differ much more than those in the gut of terrestrial animals and those that may reproduce the most efficiently and colonize the pond may simply be a matter of being in the right place at the right time. The definition of a probiotic in the case of aquaculture changes slightly though. It is still an additive of lives microbes, but rather than the result of improving the health of a host, they serve the purpose of improving the quality of the ambient environment or the water by modifying the microbial community already present. Probiotics can be used to improve the health of a host too, such as fish or other aquatic animals, but this is by changing the ambient microbial community in the case of ponds or aquaculture. For example, probiotics have been used since the 1990s in shrimp hatcheries. In this case, probiotics may also reduce disease, improve the use of feed and enhance its nutritional value. Therefore, in the pond, depending on its purpose and what resides in it, such as whether it is a decorative pond with just water or a few plants or whether it is a more natural pond with fish or other animals, the kinds of probiotics used may vary.
Since adding microbes to a pond does not directly affect a host due to the aquatic nature of the environment and serves more to alter the ambient microbial environment of a pond, the addition of probiotics may also be referred to as biocontrol. Biocontrol is the limitation or elimination of parasites or specific pathogens by the introduction of adverse microorganisms. This is often microbial treatments that target other microorganisms, like algae. Probiotics in a pond may also assist in bioremediation or bioaugmentation, when microbes are added to a pond to treat and eliminate waste and pollutants (Figure 1).

Figure 1: Biocontrol, bioremediation and bioaugmentation in a pond. Live microbial preparations, when added to a pond, can beneficially affect a host in a pond like shrimp. However, unlike the case with probiotics, this is not done by directly ingesting the probiont but rather by affecting the aquatic microbial community. So, the addition of these microbes to a pond is often referred to as biocontrol. Bioremediation, or bioaugmentation, is when the microbes added improve the water quality by breaking down pollutants and waste.

There are several effects one may expect when adding microbes to a pond. Adding certain microbes may enhance decomposition of organic matter created by plants or fish among other life forms, reduce the concentration of nitrogen and phosphorous which often enhances algal growth therefore algal blooms may be stabilized, and the availability of oxygen may be increased. Nitrifying bacteria may be added to ponds to control and reduce the concentrations of ammonia or nitrite by converting these compounds to nitrate, which is less toxic for a pond. Some reports claim that many bacterial strains may be able to reduce algal populations like those of red tide plankton and cyanobacteria (described in the last post “Phycology: The Forgotten Field of Study and the Pond”).
With all the potential functions of biocontrol in ponds, many types of preparations exist. One of the most common preparations is live bacteria supplemented with yeast extracts and extracellular enzymes in order to ensure plenty of nutrients for the bacteria and host, if necessary. Some preparations contain only the enzymes, while others contain extracts of plants such as citrus seed and yucca extracts. These preparations generally have to be added at regular intervals though because a pond already carries a well established or stable microbial community. Adding the preparation on a regular basis ensures the desired microbe’s continual dominance over others. It is also unlikely that a single bacterial species will become dominant unless added continuously in huge quantities; therefore, many preparations have several bacterial species in order to better ensure that the microbial community of choice will remain dominant.
Probiotics, biocontrol, and bioremediation are all attractive methods to improving the day to day lives of pond enthusiasts, among other people. However, there is still much to be learned about the microbial candidates and how they would produce a desired effect. Some unanswered questions include what is the best way to introduce probiotics and their optimal dose, as well as which microbes would do the best job for the specific task at hand. There are some promising candidates though, and if you speak to us at Village Pond and Garden we can determine whether or not a probiotic regimen or biocontrol is right for your pond.

In the next instalment →Physical Methods to Kill Microbes or Taking Microbes by Force

Summary/Important Points

• A probiotic is a viable mono- or mixed culture of microorganisms, which applied to animals or humans, beneficially affects the host by improving the balance of bacteria in the gut.
• A prebiotic is a non-digestable food ingredient that beneficially affects the host by selectively stimulating the growth of one or a limited number of bacteria in the gut to improve the health of the host.
• Probiotics and prebiotics can be added together in order to obtain a synergistic effect of having the selective compound and the bacterium for which it will select present. This is referred to as a symbiotic.
• Possible mechanisms for probiotics include the production of inhibitory compounds; the competition for nutrients, energy sources and adhesion sites; the alteration of the microbial metabolism, the stimulation of host immunity, the enzymatic contribution to digestion, and probiotics may be a source of macro- and micronutrients.
• In the instance of ponds, adding live microbes in order to control a microbial population is referred to as biocontrol. This is because the microbes are not directly added to the gut of the host due to the nature of the aquatic environment, and some treatments may not even target animal populations but may target other microbial populations such as algae.
• Probiotics in a pond can assist in bioremediation/bioaugmentation, which is when microbes are added in order to treat and eliminate waste and pollutants.
• In the case of ponds which generally have established microbial communities, microbe preparations will have to be added on a regular basis in order to ensure the desired microbe’s continual dominance.
• To ensure the desired continual dominance of select microbes, preparations often contain several bacterial species for biocontrol.
• There is still much to be learned about probiotics, biocontrol and bioremediation. What remains to be seen is the most efficient way to introduce them and the optimal doses. The best microbial candidates for specific tasks have yet to be determined.

References/Further Reading

Boyd, CE., Gross, A. 1998. Department of Fisheries and Allied Aquacultures Auburn University, Alabama USA 36849.
Gatesoupe, FJ. 1999. The use of probiotics in aquaculture. Aquaculture 180. 147–165.
Irianto, A., and Austin, B. 2002. Probiotics in aquaculture. Journal of Fish Diseases, 25, 633–642.
Rowland, I., Capurso, L., Collins, K., Cummings, J., Delzenne, N., Goulet, O., Guarner, F., Marteau, P., and Meier, R. 2010. Current level of consensus on probiotic science. Gut Microbes. 1(6): 436–439.
Schrezenmeir, J., and de Vrese, M. 2001. Probiotics, prebiotics, and synbiotics—approaching a definition. Am J Clin Nutr 2001;73(suppl):361S–4S.
Shanahan, F. 2010. Probiotics in Perspective. Gastroenterology. 139 (6): 1808-1812.
Verschuere, L., Rombaut,G., Sorgeloos, P., and Verstraete, W. 2000. Probiotic Bacteria as Biological Control Agents in Aquaculture. Microbiology and Molecular Biology Reviews, p. 655–671.

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Phycology: The Forgotten Field of Study and the Pond

I have revised different methods and products used to clean areas like households and ponds, as well as their advantages and disadvantages. I have also started to get into the communities of microbes that one might see in their pond, which is the biofilm that is primarily but not completely bacterial. Now I would like to go over another important group of microbes in the pond ecosystem, algae.
The study of algae is referred to as phycology. I stated in the title that it is “The Forgotten Field of Study”. I am sure there are many scientists who are quite passionate about phycology; however, I am referring to this from my point of view. I am studying cancer and, previously, have been working with bacteria and viruses. I have not studied algae since undergrad, so this is just as excellent a review for me as it is for the reader. I am quite happy to be writing about algae because these life forms are highly varied in their size, habitat, form and even method of reproduction. I found reading this topic for my blog very interesting.
The first thing one needs to know is that the word algae is plural. The singular form is alga. For example, the cyanobacteria are also known as a group as the blue-green algae. Conversely, nori is the red alga called porphyra that is eaten in sushi. Algae are a very important group of life forms. As I mentioned in my first post (Germs Are Not All Bad), algae photosynthesize and create just as much oxygen or more than all the plants in the world combined. Most algae, but not all, utilize photosynthesis to create their own nutrients. Forms of life that do this are called autotrophs, which can create organic compounds like carbohydrates, fats and proteins from inorganic compounds and light. Some algae, however, predate on other life forms for nutrients, these life forms are referred to as heterotrophs (Figure 1). Other algae, yet, are even parasites, heterotrophs that live in association with another life form and at the expense of the second life form. As for the habitat of algae most, like the algae found in ponds, live in water and form the foundations of most aquatic food chains. Algae can also live in soil, on trees, in animals as parasites, and in porous rocks like limestone and sandstone. Algae are also capable of living in varied harsh habitats, like in hot springs, polar ice and snow banks.

Figure 1: The cycle between auto- and heterotrophs. Algae, among other life forms like plants, use photosynthesis to use carbon dioxide and water to form complex organic compounds like proteins, carbohydrates and lipids with the assistance from solar energy. These compounds, in turn, are ingested by the heterotrophs for sustenance. Heterotrophs then produce water and carbon dioxide to continue the cycle.

Algae have varied habitats, but their forms are equally varied. Some of the algae are microscopic, unicellular organisms that can live alone as planktonic cells or in colonies. They can also be multicellular organisms that resemble branched and unbranched filaments, or even discs, tubes, clubs or trees. Algae can also be quite large and have highly specialized cells within them with different functions like the seaweeds which can reach 80 metres in length (Figure 2). Algae also utilize various modes of reproduction. They can reproduce asexually, sexually, and many algae actually utilize both methods of reproduction.

Figure 2: The various forms of algae. The first panel is an example of a brown alga, kelp. The second panel is the cyanobacterium called Nostoc pruniforme, which grows in large spherical colonies. The third panel is Stonewort, which is a green alga. The fourth panel is a microscopic volvox colony, another example of a green alga.
Since there exist various forms, sizes and manners in which algae reproduce, one would imagine there also are many different types of algae. The many types of algae will be enumerated and described here.

1. The cyanobacteria or blue-green algae:
These microbes are quite ubiquitous. They are found nearly everywhere in water and on land. These organisms have even been found in hot springs where the water can reach a blistering 71°C and in the crevices of desert rocks. Cyanobacteria exist as single cells, or as colonies in filaments or gelatinous masses (Figure 2). Cyanobacteria are unique among the algae because they are in the kingdom prokaryotae and are therefore prokaryotes, whereas all other algae are in the kingdom protista and are eukaryotes. Eukaryotes have organised structures called organelles within their cells. These include the mitochondria and chloroplasts, which produce energy for the cell, and the nucleus, which contains the genetic material of the cell. Prokaryotes do not have organelles. Bacteria and cyanobacteria are prokaryotes while most other life forms are eukaryotes. However, though cyanobacteria have no organelles, they do have some membranes within their cell called thylakoids, which contain chlorophyll and other components for photosynthesis. Since cyanobacteria have no organelles but are capable of photosynthesis like other algae and plants, there still exists a debate if they should be classified with bacteria or algae (Figure 3).

Figure 3: An example of the tree of life divided into bacteria and eukaryotes, and archae, which are beyond the range of the subject matter of this post and will not be covered. The kingdom prokaryotae are prokaryotes like cyanobacteria and bacteria. They have no organelle structures within their cells. The eucaryota or eukaryotes have organelles within their cells. The other algae are amongst them. There remains a debate as to where the cyanobacteria ought to be situated in the tree of life.
Another interesting aspect of cyanobacteria is that their ancestors are thought to be the earliest life forms on earth. Fossilized cyanobacteria have been found in rocks over 3 billion years old. These organisms are thought to be the first photosynthesizers which added oxygen to the atmosphere for billions of years before plants. One example of a cyanobacterium is spirulina, which is used in food because it is high in protein. Some cyanobacteria are used in rice paddies as fertilizers, while others produce toxins and in large numbers will cause swimmers’ itch.

2. The green algae:
Green algae are named so due to their photosynthetic chloroplasts which contain the green pigment chlorophyll. They can be unicellular organisms such as the phytoplankton that produce a large amount of oxygen in the atmosphere. Green algae also can be multicellular filaments which one will see in their pond as pond scum. Some of the seaweeds are amongst green algae such as Stonewort (Figure 2), which will grow several feet in length. As well as living in aquatic environments, the green algae have also been found on tree trunks and in soil.
3. The red algae:
Red algae are named so due to their red pigment called phycoerythrin, which absorbs blue light well and reflects red light to make the algae appear red. Blue light is the only light that penetrates well into deeper depths of water, so red algae that will be found in deeper depths will be almost black due to the phycoerythrin masking the pigment of chlorophyll. However, at shallower depths, the algae will appear green due to smaller proportions of phycoerythrin. These algae are mostly multicellular and will form filamentous algae and seaweed, which can be found in ponds, salt water and in damp soil though almost all species are marine algae.
Most of the algae one will see at the seashore will be red algae. One example of red algae is coralline algae. The cell walls in coralline algae become hardened with calcium carbonate, which makes up the material for coral reefs. Coincidently, I am sorting cells in my lab with a technique called flow cytometry, which identifies and sorts cells based on their fluorescent labelling. One of the fluorescent dyes I use is phycoerythrin from red algae.
4. The brown algae:
These algae are named so after their golden-brown carotenoid pigments. They are mostly unicellular and are found in lakes, ponds and oceans as phytoplankton. These algae can pose a problem to pond owners and enthusiasts because, in shallow ponds that can dry up in the summer or freeze up completely in winter, these algae can survive by forming protective cysts. One example of a brown alga is kelp, which can grow up to half a meter per day and reach up to 80m in length (Figure 2).
5. The dinoflagellates:
The dinoflagellates do not necessarily have chloroplasts to photosynthesize like most of the types of algae described earlier. In fact, many species are heterotrophs and parasites. They therefore rely on other organisms for nutrients (Figure 1). Some of these algae use harpoon-like structures called trichocysts to capture other organisms for food. Some photosynthetic species live inside invertebrate species like coral and giant clams and provide nutrients for these organisms by way of photosynthesis in a symbiotic manner while the other organism provides protection for the dinoflagellate.
A large portion of the algae are unicellular and are recognized by their strange structures such as horns, spikes and wing-like structures (Figure 4). Most of the dinoflagellates live in salt water but some can be found in fresh water like in a pond.

Figure 4: The varied forms of the microscopic dinoflagellates. The first panel is Ceratium furca, the second panel is Ceratium umitunoobimusi, and the third is an example of Pfiesteria.

An example of dinoflagellates is the red tide in which there will be a huge increase in photosynthetic dinoflagellates in coastal waters. Certain species will produce toxins which are problematic to ocean life and to consumers of seafood during these algal blooms. The red tide is named after the carotenoid pigments certain algae will possess.
One can now see how truly varied and important algae are. Algae, as alluded to throughout this post, have many uses. They are used in food such as nori, spirulina, dulse and are used as thickening agents in ice cream among many other food types. The pigments are used, for example, when I do cell sorting in the lab, as well as for dyes and colouring agents. Algae are used as fertilizers, such as the cyanobacteria mentioned with respect to rice paddies, and as livestock feed. Algae are also used as biofilters to treat waste water in order to make it fit for human use and can be used to make biodiesel fuel. On top of all their uses, environmentally speaking and for the pond enthusiast, since algae are so ubiquitous and reproduce quickly, they serve as environmental indicators in aquatic ecosystems because these organisms will be the first to change in response to an environmental change. For example, when high levels of nitrates and phosphates are found in a pond, river or lake, the algae will respond with an algal bloom, described earlier as a huge surge in the population of photosynthetic algae. If the pond or lake is small enough or if the size of the bloom is large enough, the algae may serve to detriment plants and fish also present by depleting the oxygen and light in the water.
Algae, like the microbes I described in my first post, are everywhere and as long as one has a pond, there will be algae. Even upon completely cleaning the pond and drying it out or freezing it, algae will survive. Also, algae can also spread through the air as spores. Algae, when it is in blooms, can be a really ugly sight in a pond but normal populations of algae are an essential part to the balance of the pond ecosystem. Since many types of algae can be present in a pond and will have different functions, they will need to be assessed in different ways and this can be done by contacting Village Pond and Garden if algae and the regular maintenance of a pond ecosystem is a concern. With regular maintenance of the pond with plants, bacteria, probiotics and the right cleaning regimen, we will have organisms, including algae and pond enthusiasts happy.

In the next instalment →You Get More Flies with Honey and You Get Better Microbes with Probiotics

Summary/Important Points

• Phycology is the study of algae.
• “Algae” is plural and “alga” is singular.
• Algae create just as much oxygen or more than the plants of the world by photosynthesis. Algae utilize photosynthesis to create their own nutrients, but some algae predate on other organisms for nutrients.
• Autotrophs are life forms that can create their own nutrients such as by photosynthesis, where solar energy, water and carbon dioxide are used to create complex organic compounds.
• Heterotrophs are life forms that need to obtain nutrients by ingesting other life forms.
• Algae can live in water, soil, on trees, in animals as parasites, in porous rocks, in hot springs, snow banks and polar ice. Pretty much algae can live anywhere.
• Algae exist in many forms like unicellular planktonic algae, in colonies, in multicellular filaments and as large seaweeds.
• Many types of algae exist; cyanobacteria, green algae, red algae, brown algae and dinoflagellates.
• There are many uses for algae; they are in food, their pigments are used in dyes, they can be used in fertilizers and livestock feed, and can be used as biofilters to treat waste water so it can be fit for human use once again.
• Normal populations of algae are an essential part of the pond ecosystem. Since many types of algae can exist in a pond with different functions, they need to be assessed, then maintained with plants, bacteria, probiotics and the right cleaning regimen for a specific pond.

References
http://en.wikipedia.org/wiki/File:Auto-and_heterotrophs.png
http://en.wikipedia.org/wiki/File:Kelp-forest-Monterey.jpg
http://en.wikipedia.org/wiki/File:CyanobacteriaColl1.jpg
http://en.wikipedia.org/wiki/File:CharaFragilis.jpg
http://en.wikipedia.org/wiki/File:Alga_volvox.png
http://en.wikipedia.org/wiki/File:Phylogenetic_tree.svg
http://en.wikipedia.org/wiki/File:Ceratium_furca.jpg
http://en.wikipedia.org/wiki/File:Ceratium_sp_umitunoobimusi.jpg
http://en.wikipedia.org/wiki/File:Pfiesteria_large.jpg

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How to Properly Use a Cleaner – Things One Should Know About Disinfectants

Now that I have discussed microbes and how they can be killed or have their growth stopped with the use of specific and non-specific compounds, and the types of communities microbes will form in a pond (no microbe is an island – biofilms), I would like to elaborate on cleaning and how it is important to know all about a chosen product or chemical and goal when wanting to clean. This applies to all situations in which you may want to lessen the number of microbes and thus clean a surface or area.
First, there are three large terms used for cleaners that should be clarified. It is important to know what these terms mean when selecting the proper product for the job. The first term used is sterile, or to sterilize. This is the most severe form of ridding microbes. It is actually when all microbes and life are removed and killed. All the cleaners you will buy cannot accomplish this task (as I mentioned in my first post, it is nearly impossible to remove all microbes and it is unnecessary). Only special facilities are equipped to do this, such as hospitals and labs like the one I work in. Only when doing sensitive experiments in biology or science and when using tools to treat illness, like performing surgery, is it even worth considering obtaining sterile tools and conditions. Otherwise, when considering all other aims, like household cleaning and pond maintenance, the aim is disinfection. Disinfection, or a disinfectant, is the second term. To disinfect is not to remove all microbes but to remove many microbes that may cause a problem. In fact, many commercially available disinfectants, as determined by standard testing methods, will be able to reduce microbial contamination by several orders of magnitude. The third term is an antiseptic, these are like disinfectants, but they are substances that are used on living tissue to remove problem causing microbes. One example of these is iodine when it is applied to the skin to reduce infection in an injury. All antiseptics disinfect, but not all disinfectants are antiseptic. Some disinfectants are very toxic and, therefore, you would never want to apply it to living tissue like your skin.
The disinfectants exist in a wide array of groups on which I will elaborate. Depending on the goals set out for cleaning, the choice of a disinfectant may differ. Disinfectants differ in their cost, toxicity, how quickly they break down, what sort of surfaces a disinfectant may best clean and how effectively they kill certain microbes. Disinfectants are not selective like the antimicrobials (I went over this in the first post) but, depending on how you use them, they may be more effective at killing a specific type of microbe.

1. The alcohols (ex: ethanol): These can be used as antiseptics as well. They are non corrosive but may be a fire hazard, since they are highly flammable. It can be difficult to get the full disinfectant efficacy because alcohols evaporate quickly. In other words, they have a limited residual activity. It is important to remove excess biological material (organic material) before using these disinfectants because their presence will limit the activity of the disinfectant. Higher concentrations are not necessarily the best because the alcohols diffuse better through cell membranes diluted to 70% with water, though higher concentrations have been shown to work better with certain kinds of viruses.
2. The aldehydes (ex: formaldehyde and gluteraldehyde): Some bacteria have the ability to form an extremely robust form of protection against the environment called a spore. A lot of disinfectants are useless against bacterial spores. The aldehydes have a wide spectrum of microbes they can kill including bacterial spores and fungi. Disadvantages include that they are quite toxic, partially inactivated by organic material and only have slight residual activity.
3. The phenolics (ex: phenol): This disinfectant group contains the first disinfectant discovered by Joseph Lister in 1867 and was employed first in surgeries. This disinfectant is known as phenol, or was then known as carbolic acid. The disadvantage to this disinfectant is that it is corrosive to skin and can react with some plastics. The advantage to these disinfectants is that they have a broad range of microbes that they can kill.
4. The quaternary ammonium compounds: These compounds can kill algae (algaecide) but have a short 24 hour life span until the compounds break down and are no longer effective.
5. The silver compounds: Depending on the pH in which the compounds are being used, they can last several weeks. These compounds are capable of killing algae and bacteria. One disadvantage is that if the compounds are exposed to a high pH and sunlight, they may stain some surfaces.
6. The oxidizing agents (ex: bleach, chlorine, hydrogen peroxide, iodine, ozone): These compounds include several disinfectants that are quite common in everyday usage and have many different ways in which to be effective, so I will discuss them in a little more detail. Some of these compounds, such as iodine and hydrogen peroxide, are safe enough to use on the skin as antiseptics. While others, like sodium hypochlorite or bleach, are caustic to the skin, lungs and eyes but, when diluted enough, is safe enough to be in our drinking water. Therefore, bleach especially demonstrates how, depending on the situation, a disinfectant may produce different results. At the low concentrations in drinking water, however, it is not effective at killing algae. At higher concentrations, like a 5% solution, it is effective against many common pathogens including M. tuberculosis (the bacterium that causes tuberculosis), hepatitis B and C (viruses that cause hepatitis), and, in this case, will be effective against algae. Bleach acts by attacking lipids (fats) in cell walls and then proceeds to destroy enzymes in the cell by oxidation reactions, which are defined as chemical reactions where there is a loss of electrons. The compound of sodium hypochlorite in bleach is not a good disinfectant. It must be dissolved in water to form the product called hypochlorous acid (HClO), which is the true disinfectant.

Figure 1: The dissolving of sodium hypochlorite (NaClO) in water (H2O) to create the disinfectant called hypochlorous acid (HClO) and sodium (Na+) and hydroxide (OH-) ions. The arrow indicates the chemical reaction between the reagents of sodium hypochlorite and water to create the two products of hypochlorous acid and the sodium and hydroxide ions.

NaClO+H2O → HClO+Na++OH-

Bleach is also good because it is relatively safe to use in the environment. This is because it will quickly break down into salt (NaCl) and oxygen (O2), which are not harmful. The disaavantages of bleach are that it requires 30 minutes of exposure to kill bacteria and if it is mixed with ammonia or any other acid, such as vinegar, it will cause harmful gases to form. Bleach, like other disinfectants, is less effective if organic material is present.

The overall message to be taken here is that there are a variety of disinfectants that can clean your home or pond but, depending on each situation, some cleaners will work better than others. Even the manner in which a single type of disinfectant is used may help or hinder its effectiveness. Important factors to consider are the mode of action of a certain type of disinfectant and its spectrum of activity against different types of microbes. With that known, then one must consider what types of microbes pose the problem and need to be reduced in number. In the case of ponds, the problem microbe will often be algae, which I will discuss in my fourth post (Phycology: The Forgotten Field of study and the Pond). Then one must employ the disinfectant in a fashion that assures will maximize the benefits of the compound, which include a proper concentration, time of exposure and the right surfaces and environments which will pose no problem to the use of said compound. An example of this is that some disinfectants are corrosive to certain surfaces. Here at Village Pond and Garden, if you have a pond that is not looking its cleanest, we will employ various techniques to see which microbes are causing the problem and how to best minimize their numbers by verifying the environment of the pond and by employing disinfectants, if necessary, in a way catered to your pond. Once all of the variables in a home or pond are known, then disinfecting agents can be used to their maximum potential.

Summary/Important Points

• There exist three terms that globally encompass types of cleaning. To sterilize, disinfect and an antiseptic.
• Sterilize is to completely kill all microbes. Disinfect is to greatly reduce the number of problem microbes. Antiseptics are disinfectants used on living tissue.
• Disinfectants exist in a wide array of groups, which differ in their mode of action and spectra of microbes to which they are effective (alcohols, aldehydes, phenolics, quaternary ammonium compounds, silver compounds, and oxidizing agents).
• Due to the differences in disinfectants, it is important to choose the correct one that will have the most benefits and least drawbacks for the specific job at hand.
• Factors that may affect a cleaning job and choice of disinfectants include cost, toxicity, how quickly they break down or evaporate (residual activity), which surfaces are best suited for a certain disinfectant, concentration and time of contact.
• Disinfectants can be used to their maximum potential once all variables in cleaning a home and/or pond are known.

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No Microbe is an Island: Biofilms

No Microbe is an Island: Biofilms

As I mentioned in my previous post, no microbe is an island, microbes are everywhere and will always be cohabitating with other microbes, be it with similar microbes as with a bacterium with a different species of bacterium, or with completely different microbes as with a bacterium and a virus. This is seen everywhere in our life filled world where different animals and life forms must live in the same habitat all the time.
The prevailing microbial lifestyle is for microbes to form biofilms. These are often composed of bacteria in a multispecies community, but can also be composed of other types of microbes. The bacterial biofilms are the most well known and studied, so in this post, from here on I will refer to the bacterial biofilm. In a biofilm, the bacteria will live together, stay or leave with purpose, communicate and share information in response to their environment and fill distinct niches within the community to the benefit of the bacteria involved. A biofilm is often compared to a city where people live in dense populations, share resources, communicate and have different jobs, all of which keep the city and its members functioning.
A biofilm is a microbial city, but to our naked eyes, these microscopic organisms appear as a coating or sludge when they are formed in a biofilm. Some biofilms include the plaque on our teeth, they are on the hulls of ships, on the inside of pipes, can form on indwelling devices in hospital patients and, of course, include pond scum on the surfaces of ponds such as on rocks. There are a huge number of different compositions of biofilms depending on the environment. One interesting example is the bacteria in a biofilm that developed a beneficial relationship with ants. This type of bacteria are called Actinobacteria, they are provided protection by the ants and in return will help maintain pathogen free fungal gardens for the ants (Currie 2001).

Figure 1: The highly structured biofilm formed by the bacteria Bacillus subtillus on the surface of a liquid in a beaker on the left and as a colony on the right.

A image of bateria biofilm

The formation of a biofilm amongst bacteria is a complex series of events. First, the bacterium will slow down as it approaches a surface and it will form a transient association allowing it a chance to search for a place to settle down. Then, it will migrate to other bacteria and will form a more stable attachment to the surface amongst these other bacteria. This is when the bacteria form a very small colony or a microcolony to start the formation of the biofilm. This step in the process is likened to the bacterium choosing a neighbourhood in which to live. Finally, the buildings of the microbial city go up. These are made by the bacteria and are formed from complex sugars. The sugars making the buildings in the microbial cities are the reason why biofilms often seem slimy. Sugars allow for the retention of water. Once a biofilm is formed, bacteria may stay or leave according to their particular conditions. They may leave to start up other biofilms, or may even move within the biofilm much as commuters will amongst a city.

Figure 2: The formation of a biofilm. In the first cell the bacterium initially approaches a surface as a free swimming bacterium. Then, in the second cell, the bacterium slows its motility and forms a transient association with the surface. In the third cell, the bacterium will aggregate with other bacteria and form a microcolony. In the fourth cell, the bacteria form the biofilm structures from complex sugars they produce. In the final cell, a bacterium may detach from a biofilm to move and form other biofilms.

image of the development of a biofilm

As I mentioned earlier, there are advantages for bacteria when in a biofilm. Since a biofilm constitutes a large number of bacteria and complex structures formed from sugars, biofilms offer protection and insulation from the environment. There is protection in numbers and in the biofilm structures. The structures will insulate the bacteria from getting swept away to a less favourable environment as well as prevent the diffusion of harmful substances to the bacteria from outside. For example, it is well known that bacteria in biofilms are more resistant to antibiotics, chlorine and detergents. Actually, many scientists believe that many of the antimicrobials such as antibiotics were made in response to the competition of microbes in the environment when they are amongst biofilms (Durrett and Levin, 1997). Antimicrobials prevent some microbes from entering or establishing a biofilm while giving the advantage to the microbes producing these products to colonize. As I mentioned before, in the microbial city of the biofilm, communication occurs frequently as with members of any city. This is no exception to the biofilm. Bacteria will “communicate” in response to their environment by means of producing molecules that will diffuse and be absorbed by other members of the biofilm, which will in turn affect bacterial activity. One example of this communication which I described in the previous blog post is how many bacteria will transfer a genetic trait responsible for conferring resistance to certain antibiotics to other bacteria which will not present this trait. This, in the end, will allow a formerly susceptible bacterium to become resistant to the antibiotic in question and the bacterium, in turn, can transfer the resistance trait to yet more bacteria. The transfer of antibiotic resistance is known to occur frequently in biofilms. This is yet another reason to limit the usage of antimicrobials since bacteria most often exist in biofilms. Also, antimicrobials most often act on replicating bacteria and since bacteria in biofilms share limited resources, they replicate less frequently, which is another limitation of the antimicrobial when acting on biofilms. In fact, some evidence indicates that using antimicrobials may even encourage the formation of biofilms in some instances (Hoffman et al. 2005).
Even if I did recommend the usage of antimicrobials for biofilms, they are not efficacious due to the nature of these structures. Biofilms, however, when seen in your pond, can be rather unsightly. So how does one deal with biofilms? Biofilms are structures that protect bacteria from the environment and help a bacterial colony adhere to a surface. Therefore, one of the ways to remove a biofilm is to remove the moisture and employ good old elbow grease to clean them off. Proper amounts of bleach and detergents must be used for proper amounts of time since bacteria in a biofilm tend to be harder to kill with these products (This will be elaborated in my next post “How to Properly Use a Cleaner”) This will ensure maximum results in combination with scrubbing. In the first post, there was mention of microbes, when left to their own devices, being able to form a proper ecosystem or environment in a pond. This applies to biofilms too, and we at Village Pond and Garden can help this along. We employ the use of probiotics to ensure proper microbial growth in a pond. That is, in order to limit the formation of biofilms and to ensure that bacteria better suited for the healthy ecology of a pond predominate in a biofilm that does form. We add beneficial bacteria to outnumber the bad ones and only certain sugars and metabolites that will ensure the growth of good bacteria in your pond.

Summary/Important Points

• The prevailing lifestyle of microbes is to form biofilms. These are like microbial cities, but appear to us often as a slimy film.
• Biofilms are diverse and ubiquitous. They form on our teeth (plaque), on the hulls of ships, in pipes, on indwelling devices in hospital patients, and include pond scum.
• The formation of a biofilm is a complex series of events involving the aggregation of bacteria into microcolonies and the formation of the biofilm structures out of complex sugars.
• Biofilms offer many advantages to the microbes that comprise it. It offers protection from being swept away from the surface of attachment, and from harmful substances such as antibiotics, chlorine and detergents. Biofilms also offer the opportunity to exchange information with regards to its environment to assist in microbial adaptation.
• One such communication/transfer that takes place in biofilms is the transmission of traits responsible for antibiotic resistance. Therefore, the spread of antibiotic resistance can occur in biofilms.
• The best way to deal with biofilms is not to use antimicrobials but to employ the right amount of cleaner for the right amount of time. Probiotics are also useful because, by encouraging only the growth of some bacteria, they limit the formation of biofilms and ensure that the biofilms that do form are comprised of microbes that are good for your pond.

References/Further Reading

Currie CR. 2001. A community of ants, fungi, and bacteria: A multilateral approach to studying symbiosis. Annu Rev Microbiol 55: 357–380.

Durrett, R., and S. Levin. 1997. Allelopathy in spatially distributed populations. J. Theor. Biol. 185:165–171.

Kolter, R. 2010. Biofilms in lab and nature: a molecular geneticist’s voyage to microbial ecology. International Microbiology. 13:1-7.

Lopez, D., Vlamakis, H., and Kolter, R. 2010. Biofilms. Cold Springs Harbor Perspectives in Biology.

Watnick, P., and Kolter, R. Biofilm, City of Microbes. Journal of Bacteriology. 182(10):2675-2679.

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Germs Are Not All Bad

Today, we often see advertisements for soaps, cleaning products, bandages, and other such things like brooms and wipes that claim to be antimicrobial or contain an antimicrobial. It immediately reminds me of what a germaphobic society we have become. Like all phobias, this is a fear that has gone too far with not enough information to guide it. But not all germs are bad, and not all antimicrobial products may be doing what you intend them to do.

First of all, what one needs to know is what is “antimicrobial”? It may mean a substance that stops the growth of germs (-static) or one that kills germs (-cidal). Second, antimicrobials may hinder germs by means of chemicals that are often not good for many living beings, including us (bleach, ammonia, alcohol, hydrogen peroxide, etc.). Germs may also be hindered by detergents, which are in most soap, or by more specialized compounds made by nature or humans to specifically target certain germs. Antimicrobials, most often though, refer to these compounds that target specific microbes such as antibiotics, antifungals, antiparasitics and antivirals.

The second thing we ought to know is what is a germ? As a microbiologist, I prefer the term microbe and will substitute it for germ from here on. Microbes are tiny life forms. Usually they are microscopic and cannot be seen by the naked eye unless they aggregate into large colonies. Microbes come in a plethora of forms as varied as the colours of the rainbow that can do many things, good and bad. There is the protist, which includes the algae in your pond. These microbes are responsible for putting just as much oxygen in the atmosphere as all the trees in the world. There are the fungi, which include the yeast that help us make our breads and beers. There are viruses, which include the pesky common cold and other viruses that may yet prove useful in making vaccines. Lastly are the bacteria, which include E. coli, which is necessary in our digestive tract and helps us produce vitamin K but on the flip side can cause enterohemorrhagic (bloody) diarrhoea.

 

Figure 1:  Microbes such as the amoeba (protist), E. coli (bacteria), influenza (virus) and yeast (fungi). The sizes are indicated as micrometers (um), which is 0.0001 of a centimetre. The largest microbe shown being the amoeba and the smallest here is the influenza virus.

Image of Amoeba, E. coli, Influenza and Yeast

 

So some microbes are extremely beneficial to us while others will annoy or kill us. When should we act against a microbe? It is a difficult question to answer but one logical answer would be only when a microbe may make us sick. This answer, surprisingly, is not universally accepted because some microbes are resistant to some antimicrobials such as antibiotics. So what? Treating them may not always work, and since bacteria and all microbes are everywhere and in mixed populations (no man or microbe is an island, more on this later), one may actually affect a population of microbes that was not intended to be affected or treated. When one employs an inefficacious antibiotic, it has been shown many times that a population treated will select for bacteria resistant to the antibiotic. This is called natural selection and was introduced with much controversy by Charles Darwin. Also, it is very possible for the remaining resistant bacteria to have the capacity to transfer their resistance genes or traits to other types of bacteria! This ability is unique to the microbe world but, if you were to imagine this in people, imagine that there was a gene that allowed you to become stronger and build more muscle. If you had sex with another person, rather than reproducing, this person would become larger and stronger like you. You may have heard about this with the “superbugs” in hospitals, which over time have collected so many resistance genes that they are virtually untreatable with antibiotics. This is scary because this may revert us to a time like that when we as humankind did not have antibiotics available to treat disease.

 

Figure 2: Bacterial conjugation or the ability of bacteria to transfer traits of resistance to antibiotics, as represented as the small red circle, from one bacterium to another. The bacterium on the left originally has the resistance trait and then transfers the resistance trait of the red circle to the second bacterium on the right allowing for both bacteria to be resistant.

Sketch of Conjugative Plasmids

How does one control or stop the impending takeover from the “superbug”? This returns us to the question of when we should act against a microbe. Even this is difficult to answer in the medical community where lives are at stake. In relation to this, this is why you may have heard that if you are prescribed antibiotics you should take them at the prescribed time and until the last pill is gone, even if the symptoms were gone much earlier (I implore you!). This is to maximize the chance of killing all the bacteria and allowing no resistant bacteria to survive. With regards to cleaning and ponds, I think the answer is much easier because it is not a life or death scenario. When household cleaning is involved, one only need remove dirt and grime. Microbes are everywhere and it is impossible for household cleaners to kill all germs and, even if they did, I guarantee you that the microbes will return in a few minutes. In that case, why even worry about whether one should use an antimicrobial? It will not make your house any cleaner and it may only serve to allow resistance to spread amongst the populations of microbes. We will then all be sorry when this resistance finally makes its way into a microbe that will cause us disease.

The same principals apply to ponds, which are part of our environment and contain many sorts of microbes. Actually, most microbes in your pond, when given time, will try to establish an ecosystem which will maintain itself. However, because of the scale of most garden ponds they need help. When you have a small volume of water, even as much as 7500 litres the variables tend to act and react very quickly unlike a large natural pond or lake. For example, a rapid increase or decrease in water temperature that is common in small ponds can cause the ecosystem to become unbalanced. Although this is generally not a real problem in a natural pond as it will right itself over time, in a decorative garden pond it tends to make the pond look unsightly. A spike in the water temperature can cause a bloom of algae that makes your pond look like pea soup. In a small pond it is possible that clear water may not ever be achieved without some help. I am personally in favour of probiotics, which instead of trying to kill a specific microbe will selectively promote the growth of beneficial microbes to control the overall microbe population. Our maintenance programs, designed specifically to each pond we work with, often employ a probiotic program to keep your pond clean and healthy without the use of damaging antimicrobials or chemicals.

In the next instalment → No Microbe is an Island, or the Biofilm.

Summary/Important Points

  • Not all germs are bad! Some microbes make as much oxygen as all the trees in the world, some help make bread and beer, while others are essential to our own digestion of food.
  • Antimicrobials are specialized to stop the growth or reproduction of microbes, or to kill them. These include the antibiotics, antifungals, antiparasitics and antivirals.
  • Germs are small living things called microbes, which exist in many varied forms (protists, mycoses, viruses, bacteria).
  • Microbes like bacteria can develop resistance to antibiotics and can spread this resistance to other types of bacteria.
  • In household cleaning and with ponds, products advertised to be antimicrobial should not be used. It is just recommended to use probiotics.
  • Village Pond and Garden will design an environmentally responsible maintenance program and schedule specifically for you pond.

Credits

Figure 1
Amoeba photo: Wikimedia Commons
E. coli photo: Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU. Wikimedia Commons
Influenza photo: Wikimedia Commons
Yeast photo: Frankie Robertson, Wikimedia Commons

Figure 2
Bacterial Conjugation sketch: Magnus Manske, Wikimedia Commons

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