The Effects of pH on Microbial Growth

Learning Objectives

• Illustrate and briefly describe minimum, optimum, and maximum pH requirements for growth

• Identify and describe the different categories of microbes with pH requirements for growth: acidophiles, neutrophiles, and alkaliphiles

• Give examples of microorganisms for each category of pH requirement

Yogurt, pickles, sauerkraut, and lime-seasoned dishes all owe their tangy taste to a high acid content (Figure 1). Recall that acidity is a function of the concentration of hydrogen ions [H⁺] and is measured as pH. Environments with pH values below 7.0 are considered acidic, whereas those with pH values above 7.0 are considered basic. Extreme pH affects the structure of all macromolecules. The hydrogen bonds holding together strands of DNA break up at high pH. Lipids are hydrolyzed by an extremely basic pH. The proton motive force responsible for production of ATP in cellular respiration depends on the concentration gradient of H⁺ across the plasma membrane (see Cellular Respiration). If H⁺ ions are neutralized by hydroxide ions, the concentration gradient collapses and impairs energy production. But the component most sensitive to pH in the cell is its workhorse, the protein. Moderate changes in pH modify the ionization of amino-acid functional groups and disrupt hydrogen bonding, which, in turn, promotes changes in the folding of the molecule, promoting denaturation and destroying activity. Figure 1. Lactic acid bacteria that ferment milk into yogurt or transform vegetables in pickles thrive at a pH close to 4.0. Sauerkraut and dishes such as pico de gallo owe their tangy flavor to their acidity. Acidic foods have been a mainstay of the human diet for centuries, partly because most microbes that cause food spoilage grow best at a near neutral pH and do not tolerate acidity well. (credit “yogurt”: modification of work by “nina.jsc”/Flickr; credit “pickles”: modification of work by Noah Sussman; credit “sauerkraut”: modification of work by Jesse LaBuff; credit “pico de gallo”: modification of work by “regan76″/Flickr) The optimum growth pH is the most favorable pH for the growth of an organism. The lowest pH value that an organism can tolerate is called the minimum growth pH and the highest pH is the maximum growth pH . These values can cover a wide range, which is important for the preservation of food and to microorganisms’ survival in the stomach. For example, the optimum growth pH of Salmonella spp. is 7.0–7.5, but the minimum growth pH is closer to 4.2. Figure 2. The curves show the approximate pH ranges for the growth of the different classes of pH-specific prokaryotes. Each curve has an optimal pH and extreme pH values at which growth is much reduced. Most bacteria are neutrophiles and grow best at near-neutral pH (center curve). Acidophiles have optimal growth at pH values near 3 and alkaliphiles have optimal growth at pH values above 9. Most bacteria are neutrophiles , meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7 (see Figure 2). Most familiar bacteria, like Escherichia coli , staphylococci, and Salmonella spp. are neutrophiles and do not fare well in the acidic pH of the stomach. However, there are pathogenic strains of E. coli, S. typhi, and other species of intestinal pathogens that are much more resistant to stomach acid. In comparison, fungi thrive at slightly acidic pH values of 5.0–6.0. Microorganisms that grow optimally at pH less than 5.55 are called acidophiles . For example, the sulfur-oxidizing Sulfolobus spp. isolated from sulfur mud fields and hot springs in Yellowstone National Park are extreme acidophiles. These archaea survive at pH values of 2.5–3.5. Species of the archaean genus Ferroplasma live in acid mine drainage at pH values of 0–2.9. Lactobacillus bacteria, which are an important part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5–6.8 and also contribute to the acidity of the vagina (pH of 4, except at the onset of menstruation) through their metabolic production of lactic acid. The vagina’s acidity plays an important role in inhibiting other microbes that are less tolerant of acidity. Acidophilic microorganisms display a number of adaptations to survive in strong acidic environments. For example, proteins show increased negative surface charge that stabilizes them at low pH. Pumps actively eject H⁺ ions out of the cells. The changes in the composition of membrane phospholipids probably reflect the need to maintain membrane fluidity at low pH. At the other end of the spectrum are alkaliphiles , microorganisms that grow best at pH between 8.0 and 10.5. Vibrio cholerae , the pathogenic agent of cholera , grows best at the slightly basic pH of 8.0; it can survive pH values of 11.0 but is inactivated by the acid of the stomach. When it comes to survival at high pH, the bright pink archaean Natronobacterium , found in the soda lakes of the African Rift Valley, may hold the record at a pH of 10.5 (Figure 3). Extreme alkaliphiles have adapted to their harsh environment through evolutionary modification of lipid and protein structure and compensatory mechanisms to maintain the proton motive force in an alkaline environment. For example, the alkaliphile Bacillus firmus derives the energy for transport reactions and motility from a Na⁺ ion gradient rather than a proton motive force. Many enzymes from alkaliphiles have a higher isoelectric point, due to an increase in the number of basic amino acids, than homologous enzymes from neutrophiles. Figure 3. View from space of Lake Natron in Tanzania. The pink color is due to the pigmentation of the extreme alkaliphilic and halophilic microbes that colonize the lake. (credit: NASA)

Survival at the Low pH of the Stomach

Peptic ulcers (or stomach ulcers ) are painful sores on the stomach lining. Until the 1980s, they were believed to be caused by spicy foods, stress, or a combination of both. Patients were typically advised to eat bland foods, take anti-acid medications, and avoid stress. These remedies were not particularly effective, and the condition often recurred. This all changed dramatically when the real cause of most peptic ulcers was discovered to be a slim, corkscrew-shaped bacterium, Helicobacter pylori . This organism was identified and isolated by Barry Marshall and Robin Warren, whose discovery earned them the Nobel Prize in Medicine in 2005. The ability of H. pylori to survive the low pH of the stomach would seem to suggest that it is an extreme acidophile. As it turns out, this is not the case. In fact, H. pylori is a neutrophile. So, how does it survive in the stomach? Remarkably, H. pylori creates a microenvironment in which the pH is nearly neutral. It achieves this by producing large amounts of the enzyme urease, which breaks down urea to form NH₄⁺ and CO₂. The ammonium ion raises the pH of the immediate environment. This metabolic capability of H. pylori is the basis of an accurate, noninvasive test for infection. The patient is given a solution of urea containing radioactively labeled carbon atoms. If H. pylori is present in the stomach, it will rapidly break down the urea, producing radioactive CO₂ that can be detected in the patient’s breath. Because peptic ulcers may lead to gastric cancer, patients who are determined to have H. pylori infections are treated with antibiotics.

Temperature and Microbial Growth

Learning Objectives

• Illustrate and briefly describe minimum, optimum, and maximum temperature requirements for growth

• Identify and describe different categories of microbes with temperature requirements for growth: psychrophile, psychrotrophs, mesophile, thermophile, hyperthermophile

• Give examples of microorganisms in each category of temperature tolerance

When the exploration of Lake Whillans started in Antarctica, researchers did not expect to find much life. Constant subzero temperatures and lack of obvious sources of nutrients did not seem to be conditions that would support a thriving ecosystem. To their surprise, the samples retrieved from the lake showed abundant microbial life. In a different but equally harsh setting, bacteria grow at the bottom of the ocean in sea vents, where temperatures can reach 340 °C (700 °F). Microbes can be roughly classified according to the range of temperature at which they can grow. The growth rates are the highest at the optimum growth temperature for the organism. The lowest temperature at which the organism can survive and replicate is its minimum growth temperature . The highest temperature at which growth can occur is its maximum growth temperature . The following ranges of permissive growth temperatures are approximate only and can vary according to other environmental factors. Organisms categorized as mesophiles (“middle loving”) are adapted to moderate temperatures, with optimal growth temperatures ranging from room temperature (about 20 °C) to about 45 °C. As would be expected from the core temperature of the human body, 37 °C (98.6 °F), normal human microbiota and pathogens (e.g., E. coli, Salmonella spp., and Lactobacillus spp.) are mesophiles. Organisms called psychrotrophs , also known as psychrotolerant, prefer cooler environments, from a high temperature of 25 °C to refrigeration temperature about 4 °C. They are found in many natural environments in temperate climates. They are also responsible for the spoilage of refrigerated food.

Clinical Focus: Nataliya, Resolution

This example concludes Nataliya’s story that started in How Microbes Grow and Oxygen Requirements for Microbial Growth. The presence of Listeria in Nataliya’s blood suggests that her symptoms are due to listeriosis , an infection caused by L. monocytogenes. Listeriosis is a serious infection with a 20% mortality rate and is a particular risk to Nataliya’s fetus. A sample from the amniotic fluid cultured for the presence of Listeria gave negative results. Because the absence of organisms does not rule out the possibility of infection, a molecular test based on the nucleic acid amplification of the 16S ribosomal RNA of Listeria was performed to confirm that no bacteria crossed the placenta. Fortunately, the results from the molecular test were also negative. Nataliya was admitted to the hospital for treatment and recovery. She received a high dose of two antibiotics intravenously for 2 weeks. The preferred drugs for the treatment of listeriosis are ampicillin or penicillin G with an aminoglycoside antibiotic. Resistance to common antibiotics is still rare in Listeria and antibiotic treatment is usually successful. She was released to home care after a week and fully recovered from her infection. L. monocytogenes is a gram-positive short rod found in soil, water, and food. It is classified as a psychrophile and is halotolerant. Its ability to multiply at refrigeration temperatures (4–10 °C) and its tolerance for high concentrations of salt (up to 10% sodium chloride [NaCl]) make it a frequent source of food poisoning. Because Listeria can infect animals, it often contaminates food such as meat, fish, or dairy products. Contamination of commercial foods can often be traced to persistent biofilms that form on manufacturing equipment that is not sufficiently cleaned. Listeria infection is relatively common among pregnant women because the elevated levels of progesterone downregulate the immune system, making them more vulnerable to infection. The pathogen can cross the placenta and infect the fetus, often resulting in miscarriage, stillbirth, or fatal neonatal infection. Pregnant women are thus advised to avoid consumption of soft cheeses, refrigerated cold cuts, smoked seafood, and unpasteurized dairy products. Because Listeria bacteria can easily be confused with diphtheroids, another common group of gram-positive rods, it is important to alert the laboratory when listeriosis is suspected. The organisms retrieved from arctic lakes such as Lake Whillans are considered extreme psychrophiles (cold loving). Psychrophiles are microorganisms that can grow at 0 °C and below, have an optimum growth temperature close to 15 °C, and usually do not survive at temperatures above 20 °C. They are found in permanently cold environments such as the deep waters of the oceans. Because they are active at low temperature, psychrophiles and psychrotrophs are important decomposers in cold climates. Figure 1. A black smoker at the bottom of the ocean belches hot, chemical-rich water, and heats the surrounding waters. Sea vents provide an extreme environment that is nonetheless teeming with macroscopic life (the red tubeworms) supported by an abundant microbial ecosystem. (credit: NOAA) Organisms that grow at optimum temperatures of 50 °C to a maximum of 80 °C are called thermophiles (“heat loving”). They do not multiply at room temperature. Thermophiles are widely distributed in hot springs, geothermal soils, and manmade environments such as garden compost piles where the microbes break down kitchen scraps and vegetal material. Examples of thermophiles include Thermus aquaticus and Geobacillus spp. Higher up on the extreme temperature scale we find the hyperthermophiles , which are characterized by growth ranges from 80 °C to a maximum of 110 °C, with some extreme examples that survive temperatures above 121 °C, the average temperature of an autoclave. The hydrothermal vents at the bottom of the ocean are a prime example of extreme environments, with temperatures reaching an estimated 340 °C (Figure 1). Microbes isolated from the vents achieve optimal growth at temperatures higher than 100 °C. Noteworthy examples are Pyrobolus and Pyrodictium , archaea that grow at 105 °C and survive autoclaving. Figure 2 shows the typical skewed curves of temperature-dependent growth for the categories of microorganisms we have discussed. Figure 2. The graph shows growth rate of bacteria as a function of temperature. Notice that the curves are skewed toward the optimum temperature. The skewing of the growth curve is thought to reflect the rapid denaturation of proteins as the temperature rises past the optimum for growth of the microorganism. Life in extreme environments raises fascinating questions about the adaptation of macromolecules and metabolic processes. Very low temperatures affect cells in many ways. Membranes lose their fluidity and are damaged by ice crystal formation. Chemical reactions and diffusion slow considerably. Proteins become too rigid to catalyze reactions and may undergo denaturation. At the opposite end of the temperature spectrum, heat denatures proteins and nucleic acids. Increased fluidity impairs metabolic processes in membranes. Some of the practical applications of the destructive effects of heat on microbes are sterilization by steam, pasteurization, and incineration of inoculating loops. Proteins in psychrophiles are, in general, rich in hydrophobic residues, display an increase in flexibility, and have a lower number of secondary stabilizing bonds when compared with homologous proteins from mesophiles. Antifreeze proteins and solutes that decrease the freezing temperature of the cytoplasm are common. The lipids in the membranes tend to be unsaturated to increase fluidity. Growth rates are much slower than those encountered at moderate temperatures. Under appropriate conditions, mesophiles and even thermophiles can survive freezing. Liquid cultures of bacteria are mixed with sterile glycerol solutions and frozen to −80 °C for long-term storage as stocks. Cultures can withstand freeze drying (lyophilization) and then be stored as powders in sealed ampules to be reconstituted with broth when needed. Macromolecules in thermophiles and hyperthermophiles show some notable structural differences from what is observed in the mesophiles. The ratio of saturated to polyunsaturated lipids increases to limit the fluidity of the cell membranes. Their DNA sequences show a higher proportion of guanine–cytosine nitrogenous bases, which are held together by three hydrogen bonds in contrast to adenine and thymine, which are connected in the double helix by two hydrogen bonds. Additional secondary ionic and covalent bonds, as well as the replacement of key amino acids to stabilize folding, contribute to the resistance of proteins to denaturation. The so-called thermoenzymes purified from thermophiles have important practical applications. For example, amplification of nucleic acids in the polymerase chain reaction (PCR) depends on the thermal stability of Taq polymerase , an enzyme isolated from T. aquaticus. Degradation enzymes from thermophiles are added as ingredients in hot-water detergents, increasing their effectiveness.

Other Environmental Conditions that Affect Growth

Learning Objectives

• Identify and describe different categories of microbes with specific growth requirements other than oxygen, pH, and temperature, such as altered barometric pressure, osmotic pressure, humidity, and light

• Give at least one example microorganism for each category of growth requirement

Microorganisms interact with their environment along more dimensions than pH, temperature, and free oxygen levels, although these factors require significant adaptations. We also find microorganisms adapted to varying levels of salinity, barometric pressure, humidity, and light.

Osmotic and Barometric Pressure

Figure 1. Photograph taken from space of the Great Salt Lake in Utah. The purple color is caused by high density of the alga Dunaliella and the archaean Halobacterium spp. (credit: NASA) Most natural environments tend to have lower solute concentrations than the cytoplasm of most microorganisms. Rigid cell walls protect the cells from bursting in a dilute environment. Not much protection is available against high osmotic pressure . In this case, water, following its concentration gradient, flows out of the cell. This results in plasmolysis (the shrinking of the protoplasm away from the intact cell wall) and cell death. This fact explains why brines and layering meat and fish in salt are time-honored methods of preserving food. Microorganisms called halophiles (“salt loving”) actually require high salt concentrations for growth. These organisms are found in marine environments where salt concentrations hover at 3.5%. Extreme halophilic microorganisms, such as the red alga Dunaliella salina and the archaeal species Halobacterium in Figure 1, grow in hypersaline lakes such as the Great Salt Lake, which is 3.5–8 times saltier than the ocean, and the Dead Sea, which is 10 times saltier than the ocean. Dunaliella spp. counters the tremendous osmotic pressure of the environment with a high cytoplasmic concentration of glycerol and by actively pumping out salt ions. Halobacterium spp. accumulates large concentrations of K⁺ and other ions in its cytoplasm. Its proteins are designed for high salt concentrations and lose activity at salt concentrations below 1–2 M. Although most halotolerant organisms, for example Halomonas spp. in salt marshes, do not need high concentrations of salt for growth, they will survive and divide in the presence of high salt. Not surprisingly, the staphylococci, micrococci, and corynebacteria that colonize our skin tolerate salt in their environment. Halotolerant pathogens are an important cause of food-borne illnesses because they survive and multiply in salty food. For example, the halotolerant bacteria S. aureus, Bacillus cereus, and V. cholerae produce dangerous enterotoxins and are major causes of food poisoning. Microorganisms depend on available water to grow. Available moisture is measured as water activity (a _w ) , which is the ratio of the vapor pressure of the medium of interest to the vapor pressure of pure distilled water; therefore, the a_w of water is equal to 1.0. Bacteria require high a_w (0.97–0.99), whereas fungi can tolerate drier environments; for example, the range of a_w for growth of Aspergillus spp. is 0.8–0.75. Decreasing the water content of foods by drying, as in jerky, or through freeze-drying or by increasing osmotic pressure, as in brine and jams, are common methods of preventing spoilage. Microorganisms that require high atmospheric pressure for growth are called barophiles . The bacteria that live at the bottom of the ocean must be able to withstand great pressures. Because it is difficult to retrieve intact specimens and reproduce such growth conditions in the laboratory, the characteristics of these microorganisms are largely unknown.

Light

Photoautotrophs, such as cyanobacteria or green sulfur bacteria , and photoheterotrophs, such as purple nonsulfur bacteria , depend on sufficient light intensity at the wavelengths absorbed by their pigments to grow and multiply. Energy from light is captured by pigments and converted into chemical energy that drives carbon fixation and other metabolic processes. The portion of the electromagnetic spectrum that is absorbed by these organisms is defined as photosynthetically active radiation (PAR). It lies within the visible light spectrum ranging from 400 to 700 nanometers (nm) and extends in the near infrared for some photosynthetic bacteria. A number of accessory pigments, such as fucoxanthin in brown algae and phycobilins in cyanobacteria, widen the useful range of wavelengths for photosynthesis and compensate for the low light levels available at greater depths of water. Other microorganisms, such as the archaea of the class Halobacteria , use light energy to drive their proton and sodium pumps. The light is absorbed by a pigment protein complex called bacteriorhodopsin, which is similar to the eye pigment rhodopsin. Photosynthetic bacteria are present not only in aquatic environments but also in soil and in symbiosis with fungi in lichens. The peculiar watermelon snow is caused by a microalga Chlamydomonas nivalis , a green alga rich in a secondary red carotenoid pigment (astaxanthin) which gives the pink hue to the snow where the alga grows.

Media Used for Bacterial Growth

Learning Objectives

• Identify and describe culture media for the growth of bacteria, including examples of all-purpose media, enriched, selective, differential, defined, and enrichment media

The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.

Nutritional Requirements

The number of available media to grow bacteria is considerable. Some media are considered general all-purpose media and support growth of a large variety of organisms. A prime example of an all-purpose medium is tryptic soy broth (TSB) . Specialized media are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. One type, enriched media , contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms , organisms that cannot make certain nutrients and require them to be added to the medium. When the complete chemical composition of a medium is known, it is called a chemically defined medium . For example, in EZ medium , all individual chemical components are identified and the exact amounts of each is known. In complex media , which contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. Amounts of individual components are undetermined and variable. Nutrient broth, tryptic soy broth, and brain heart infusion , are all examples of complex media. Figure 1. On this MacConkey agar plate, the lactose-fermenter E. coli colonies are bright pink. Serratia marcescens, which does not ferment lactose, forms a cream-colored streak on the tan medium. (credit: American Society for Microbiology) Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called selective media . An example of a selective medium is MacConkey agar . It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria , particularly the Enterobacteriaceae . These species are commonly named enterics, reside in the intestine, and are adapted to the presence of bile salts. The enrichment cultures foster the preferential growth of a desired microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want to isolate bacteria that break down crude oil, hydrocarbonoclastic bacteria , sequential subculturing in a medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria. The differential media make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. Color changes are the result of end products created by interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium. In Figure 1, the differential fermentation of lactose can be observed on MacConkey agar. The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.

Soysoap, Brix and Soybeans Way North Double Crop Line

Experiment: The soybeans were planted August 15th, 2008 and were at their 6^th week at 12 inches before treatment. The Soybeans were near Lansing, Michigan in 2008. The starting location of this double crop soybean experiment was 250 miles north of I-70 so the double crop. The experiment was to see if we could double crop soybeans, and we did although we had 2 nights of 10 or more hours at 26 degrees, untreated plants all died. The test was to see how Soybeans reacted to Soysoap and Brix Levels observations. The purpose of this is to show that achieving higher Brix means higher C02 consumption for the plants, healthier plant, frost protection and increased crop production. Healthy Soybeans can defend themselves against Pests and Weather.

Brix before spraying on both plots at 11:55 tested Brix at 11 am, Temperature was 84 degrees and sunny day.

9-3-2008 2.35 PM, Brix after spraying Soysoap on Soybeans, and not spraying Soysoap on Soybeans. After just 3 hours of spraying Soysoap. Soybean Brix with Soysoap 17, Soybean Brix without Soysoap 12. Comment: Soysoaped Soybeans increased Brix levels from 11 to 17 or 54%. Physiologically the Soybean sugar factory had reacted to the Soysoap with a 54% increase in Brix, which eventually would lead us to the accidentally develop our frost protection product. The untreated soybean increased Brix only 10% which could just be attributed to natural photosynthesis process during the day as it was a sunny
day.

Date	Time	Temp	Weather	Soysoap	No Soysoap	Comments
				Brix	Brix
09/04/08	9 am	70		9	8	Day 2, To early at 9.30 to take Brix reading should be in the middle of afternoon. Brix was lower than we started, we were concerned.
font color=black size="4">09/05/08	5 pm	70	Rained 1/8	13	8	CommentsDay 3, Now that we had a proper time for Brix reading the project was looking better. Soysoaped Beans were 13 vs 8, 5 points higher.
09/06/08	10 am	65		10	8	Day 4, Another Early Brix Reading, Results obvious!
09/07/08	6 pm	68		12	8	Day 5, 4 points advantage we are seeing a pattern.
09/09/08	1 pm	63	Rained 1”	11	9	Day 7, Rainy cloudy day less Brix increase expected.

09-10-08: Comment: Phase 1 of experiment, After 7 days the farmer decided to spray Soysoap again. Farmer had taken unfortunately Brix reading inconsistently during the day from 9 am to 6 pm. But in spite of that the Soybeans with Soysoap always had higher Brix Levels everyday. Phase 2 starting comments: 09-10-08 1:00p.m. 68 degrees sunny and 10 mph wind, Soybeans with Soysoap Brix 15, Soybeans without Soysoap without Brix 15. Second foliar spray began at 1:00p.m. checked again @ 3:30p.m. Soybeans with Soysoap Brix 15, Soybeans without Soysoap Brix 12. We applied 2 oz of Soysoap with the mixture of tracite, sugar, sea salt vinegar and ammonia.

09/10/08	4 pm	68	Sunny	15	12	3 points higher, Summary of this day above!
09/11/08	2 pm	68	<	15	12	3 points higher
09/12/08	1pm	72	Cloudy/Rain	Comments8	Comments8	0 points difference
09/15/08	2 pm	61	Rain 5”	8	8	0 points difference, rain again no sun.
09/22/08	1 pm	70	No rain	7	4	3 points difference
09/25/08	1 pm	76	Sunny	8	7	1 point difference
09/27/08	1 pm	72	<	9	7	2 points difference
10/02/08	1 pm	64	Cloudy	15	12	3 points difference
10/03/08	4 pm	52	Sunny	16	14	1 point difference

FREEZE WARNING: 10-03-08 6:20p.m, 50 degrees freeze forecast, Soybeans with Soysoap Brix16, Soybeans without Soysoap without Brix 12. Sprayed the bean plants with Soysoap (8 oz) and sugar (5lbs) 13 gal H2O. Farmer called about Frost/Freeze, and we guessed and told him to use the Soysoap to activate the sugar factory and drain all the water out of the plants xylem and phloem, it worked. All untreated plants died after first freeze.

10-14-08 1:30 pm 60 degrees had 3 freezes since last Brix checks: Soybeans Soysoap Brix with 14, Soybeans without Soysoap 14 this was our last Brix checks we did do pod counts on the two plots with the following results:

Test 1 pod count with Soysoap 9-15-11-5-12=52 52./.5=10.4
Test 1 pod count without Soysoap 8-4-3-2-3=20./.5=4

Test 2 pod count with Soysoap 12-18-14-15-6=65./.5=13
Test 2 pod count without Soysoap 6-5-3-7-1=22./.5=4.4

Average pod count test with Soysoap 13+10.4=23/2 = 10.4 pods
Average pod count test without Soysoap 4.4+4 =8.4/2= 4.2 pods

Farmer Final Report: Untreated Plants No Soybeans, Soysoap Treated Beans 12 bu acre. Not Great but they grew them.

Soysoap Report: Soysoap Beans had higher Brix Levels than Untreated Every Test.

Final Tally on Brix for CO2 Consumption: Soysoaped Beans Averaged 11.75, Untreated 9.31 = Treated 21% CO2 Consumption.

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