Effects Of Ph On Rhizobium Essay, Research Paper

Is there an effect on Rhizobium bacteria by changing its pH?

Steve Lincoln

Period 3

12/7/98

Question

What are the effects of varying pH levels on Rhizobium bacteria, and can a pH resistant Rhizobium culture be grown?

Hypothesis

Alterations of the balanced habitat such as the change of pH levels can drastically alter the life of bacteria and can directly affect chemical reactions of enzymes released by the bacteria. Using this information the change in pH will have an effect on the Rhizobium bacteria.

Controls

1. Temperature

2. Light

3. Amount of Bacteria for inoculations

4. Disk size

5. Broth amount

6. Sterilization Techniques

7. Amount of agar

8. Amount of buffer solution

9. Amount of Bacteria spread

10. Benchmark of Spectrophotometer

11. Growth time allowed

12. Humidity

13. Storage of bacteria

Variable

1. The change of the pH level.

Materials List

1. Presterilized pipets

2. 10 microliter inoculation loops

3. Ethyl Alcohol

4. Distilled (De-ionized) water

5. 1000mL Beaker

6. Cuvettes

7. Spectrophotometer

8. Petri Dishes

9. Rhizobium Agar

10. Rhizobium Broth

11. 100 mL Beakers

12. French Squares

13. Heat Gun

14. Filter Paper

15. Buffer Solutions (pH 4, and pH 10)

16. Forceps

17. Autoclave

18. Incubator

19. Bacti Spreader

20. Various compounds (Specified in Procedure)

21. Rhizobium Bacteria (Catalog #85 W 1907 from Ward Natural Sciences)

Procedure

1. Prepare Rhizobium agar and sterilize. (Autoclave @ 20psi for 20 minutes)

Rhizobium Agar Preparation

Add the following ingredients in 1000mL of Distilled water. Stir thoroughly

K2HPO4 .66g CaCL2 .10g

KH2PO4 .34g CaCO3 5.00g

MgSO4 .20g Mannitol 10.0g

NaCl .10g Agar 18.0g

FeSO4 .002g Yeast Extract 1.0g

MnSO4 .002g

2. Pour agar into six petri dishes and wait until they solidify.

3. Using a sterilized pipet extract .5mL of Rhizobium culture.

Rhizobium Bacteria (Catalog #85 W 1907 from Ward Natural Sciences)

4. Drop the solution onto the center of the dish and spread gently using a Bacti spreader.

5. Repeat 3 and 4 making sure to practice proper aseptic techniques between applications.

6. Cover all dishes

7. Punch holes in filter paper. Then take the disks, put them in an autoclave-safe vial and sterilize

8. Prepare buffer solutions of pH 4 and pH 10 and water.

9. Using the forceps, soak a disk in the pH 4 solution and place almost on the rim of the dish.

10. Repeat process for 5 more disks, for a total of 6 making sure each disk is equidistant from each other.

11. Complete steps 9 and 10 for pH 10 and water.

12. Place all dishes in incubator at 25| C.

13. Let bacteria grow for at least 48 hours since they were put in the incubator.

14. Retrieve dishes from incubator and measure the diameter of the halo s around the disks for each dish.

15. Record the results.

16. Prepare 3 rhizobium broths (exactly the same as the agar, however no agar is placed into the mix.) in French squares. Using a 10-microliter-inoculation loop, scrape the inside of a few of the halo s of a dish and insert into broth and stir gently. Do this for each distinct dish. (Note: scrape the two most define halo s of a pH dish grouping, e.g. The best halos of dish 1 and 2 of pH 4)

17. Label the three squares accordingly to the bacteria put in. (The purpose of this is to determine the strength of the bacteria straight from the dishes, and to produce 3 samples in which to draw on for the next test)

18. Place petri dishes and broths in incubator. Important : Broth culture lids must be slightly cracked to allow for oxygen.

19. Let the broth cultures grow for 48 hours. Then perform a transmittance test using a spectrophotometer @ a wavelength of 525.

20. Use a sterile rhizobium broth to benchmark the machine.

21. Record the results. The test should be done 10 times for each sample, allowing the spectrophotometer to return to 0 transmittance each time.

22. Prepare 3 more broths adding 10mL of pH 4 buffer solution to one square and 8 mL of pH 10 buffer solution to another square. Sterilize all three squares.

23. Using the inoculation loop, take the pH 4 bacteria and put it into the new broth with a pH 10. Do this with the 10 into the 4. The control will be transferred to the broth that did not change.

24. Place all cultures into the incubator and repeat steps 19 and 20 for the new broths.

25. Repeat experiments for more trials.

Disposal

All glass-ware that is autoclave safe can be placed directly in the autoclave. All other items infected that are not autoclave are to be put in an autoclave bag. (Petri dishes, pipets, etc.)

A L L S T E R I L I Z A T I O N:

Set the autoclave @ 20 psi for a time period of 20 minutes.

Abstract

EFFECTS OF pH ON RHIZOBIUM BACTERIA

Lincoln, Steven W.

Pinellas Regional Science & Engineering Fair Microbiology

Rhizobium bacteria plays an important role in the development of legumes (Flowering plants). Rhizobium aids in the process of nitrogen fixation. Nitrogen, which is an element of all proteins, is extremely important. Elemental nitrogen, which makes up 80 percent of the atmosphere isn t directly available for use to plants and organisms. These plants and organisms rely on the rhizobium to produce it. Through a symbiotic relationship between legumes and Rhizobium, Nitrogen Gas (N2) is transformed into a compound which then is available for biotic use. For those plants to grow in more inhospitable regions (e.g., Acidic soil) the rhizobium must also be able survive also.

In this project Rhizobium went under testing by changing the level of pH of its surroundings to find if it could live in a basic and acidic environment. Procedures were also conducted to possibly culture a strain of Rhizobium resistant to much more harsh environments than the original bacteria that was started with. The first of these tests was a Disk Diffusion, which you spread the bacteria over a petri dish and soak a punched hole of filter paper with a pH of 4, 10, or 7(Control) using buffer solutions and water for the control. The objective of the test was to measure the halo size, or the depression of growth around the disk. This shows that the Rhizobium has problems with living in that area. After these halos are measured the bacteria is sub-cultured into a neutral broth(meaning no change in pH). This is to determine the strength of the bacteria taken from the halo s of the plate bacteria through a test using a spectrophotometer. The Rhizobium is then transferred to three other broths which have undergone pH alterations. The Rhizobium coming from pH 4 plates in the beginning are now placed into the new pH 10 broths. This tests the range of tolerance of the Rhizobium.

The results confirmed the hypothesis, which stated that the change in the living environment would have an effect on the Rhizobium. The data shows a drop in growth from the initial pH 4 disk diffusion and similar drop from the pH 10 disks, however not as significant. The ensuing spectrophotometer transmittance test confirmed this. The switch spectrophotometer trans. test where the bacteria switched pH s shows surprising results. The pH 4 bacterium that was now in the pH 10 solution was killed completely. The pH 10 bacteria that were transplanted into the pH 4 solution sustained the change very well, doing better than the control.

What does Rhizobium do?

The unique ecological role of the Fabales(flowering plants legumes) is in nitrogen fixation. Nitrogen is an element of all proteins and is an essential component in both plant and animal metabolism. Although elemental nitrogen makes up about 80 percent of the atmosphere, it is not directly available to living organisms; nitrogen that can be metabolized by living organisms must be in the form of nitrates or ammonia compounds. Through a mutual benefit arrangement (symbiosis) between legumes and Rhizobium bacteria, nitrogen gas (N2) is fixed into a compound and then becomes available to the biotic world. The legume plant furnishes a home and subsistence for the bacteria in root nodules. In a complex biosynthetic interaction between the host plant and the bacterium, nitrogen compounds are formed that are used by the host plant. These compounds are also available to other plants after decayed roots (and other plant parts) of the host plant have allowed these nitrogen products to be released into the soil. Animals obtain compound nitrogen by eating plants.

Consequently, the vegetation of the forests, prairies, and deserts of most of the world is primarily dependent on the legume component of their vegetation and could not exist without it. Only in a few ecosystems–those that include but few legume species–have substitute biological nitrogen-fixing arrangements evolved. These include symbiotic relationships between miscellaneous woody species other than legumes, and certain actinomycetes bacteria and are limited mostly to boreal evergreen forests, certain coastal areas, and acid bogs. Nitrogen fixation by free-living cyanobacteria seems to be important in aquatic ecosystems. On a worldwide scale, however, these alternate arrangements of nitrogen fixation are relatively minor compared to those supported by legumes.

Legume nitrogen fixation is also of prime importance in agriculture. Before the use of synthetic fertilizers in the industrial countries, the cultivation of crop plants, with the exception of rice, was dependent on legumes and plant and animal wastes (as manure) for nitrogen fertilization. A common procedure was the use of crop rotation usually the alternation of a cash grain crop such as corn (maize) with a legume, often alfalfa (Medicago sativa), in the temperate world. Apart from the nitrogen contribution, the legume in this case furnishes animal forage (hay or silage). Pastures or other grazing areas must have legume components, such as a clover (Trifolium), as well as a grass component.

Text from

“Angiosperms: The Flowering Plants: CLASS MAGNOLIOPSIDA: THE DICOTYLEDONS: Subclass Rosidae: REPRESENTATIVE GROUPS: Fabales.: Ecological and economic importance.” Britannica Online.

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Aseptic Technique Terminology

Antimicrobial – An agent or action that kills or inhibits the growth of micro-organisms.

Antiseptic – A chemical agent that is applied topically to inhibit the growth of micro-organisms.

Asepsis – Prevention of microbial contamination of living tissues or sterile materials by excluding, removing or killing micro-organisms.

Autoclave – A steam sterilizer consisting of a metal chamber constructed to withstand the pressure that is required to raise the temperature of steam to the level required for sterilization. Early models were termed “autoclaves” because they were fitted with a self-closing door.

Bactericide – A chemical or physical agent that kills vegetative (non-spore forming) bacteria.

Bacteriostat – An agent that prevents multiplication of bacteria.

Commensals – Non-pathogenic micro-organisms that are living and reproducing as human or animal parasites.

Contamination – Introduction of micro-organisms to sterile articles, materials or tissues.

Disinfectant – An agent that is intended to kill or remove pathogenic micro-organisms, with the exception of bacterial spores.

Pasteurization – A process that kills nonspore-forming micro-organisms by hot water or steam at 65-100oC.

Pathogenic – A species that is capable of causing disease micro-organism in a susceptible host.

Sanitization – A process that reduces microbial contamination to a low level by the use of cleaning solutions, hot water or chemical disinfectants.

Sterilant – An agent that kills all types of micro-organisms.

Sterile – Free from micro-organisms.

Sterilization – The complete destruction of micro-organisms.

Definitions from

ESSENTIALS FOR ANIMAL RESEARCH: A PRIMER FOR RESEARCH PERSONNEL

Chapter 5: Principles of Aseptic Technique

http://www.fsu.edu/ FSULAR/esasept.html

What happens during nodulation?

There are actually several different mechanisms by which rhizobia induce nodule formation. The best studied, as found in plants such as clovers, alfalfa, bean and soybean, involves rhizobial infection of developing root hairs. However, in peanuts, rhizobia gain entry to their host via wounds made as lateral roots emerge, and in this species, nodules are usually found in the angle made by the tap and lateral roots.

Root hair infection involves complicated signalling between host and rhizobia. This is necessary because most of the genes needed for nodule formation by rhizobia are only activated by the presence of a suitable host plant. Activation is achieved by the secretion of chemicals called flavonoids from the germinating root. There are a number of these substances, and different legumes tend to produce a mix of flavinoids that is unique to that host.

Rhizobia attach to still-growing root hairs in a region just behind the root tip, and fully mature root hairs are rarely infected. While in contact with their host, the rhizobia produce complex chitin-like substances termed nod factors – more technically lipo-oligosaccharides.

Irrespective of the Rhizobium involved, these substances all have a basically similar core structure, with small chemical differences in this structure determining which and how many legumes are nodulated. When the appropriate purified nod factor is applied to its host at concentrations as low as 10-9 M, nodules are formed, even in the absence of rhizobia. Presence of the rhizobia causes modification in the structure of the root hair cell wall, and permits penetration by the rhizobia. Rhizobia are never really permitted open access to the host. They are always surrounded by a plant-derived mucilaginous material and, even as they move down the root hair in the direction of the root, rhizobia elongate and continue to surround the root. The structure that results is called an infection thread.

Many infections do not develop into functional nodules. For this to occur rhizobia must contact cells in the host cortex that have been stimulated to division by the presence of the nod-factor. Rhizobia — still enclosed within a plant derived barrier that mutes possible host defence responses — are released into these cells, where their multiplication leads to the development of the typical nodule structure.

Text from:

Common Questions : What happens during nodulation? , University of Minnesota: Rhizobium Research Labatory

http://www.rhizobium.umn.edu/Commonquest/whathappens.htm

pH in relation to Bacteria

Most bacteria grow in the range of neutral pH values, between 5 and 8, although a few bacterial species have adapted to life at more acidic or alkaline extremes. For example, when coal seams are exposed to air through mining operations, the pyritic ferrous sulfide deposits are attacked by Thiobacillus ferrooxidans to generate sulfuric acid, which lowers the pH to 2.0 or even 0.7. This organism can tolerate high concentrations of iron, copper, cobalt, nickel, and zinc ions and acidity as low as pH 1.3. This acid tolerance applies only to sulfuric acid; such bacteria are killed by equivalent concentrations of other acids, such as hydrochloric acid. Because bogs, pine forests, and some lakes are fairly acidic (pH between 3.7 and 5.5) and are not inhabited by many bacteria, especially under anaerobic conditions, plant polymers degrade slowly. These locales are, however, home to several types of bacteria, all gram-negative. Alkalophilic bacteria able to grow in alkaline concentration as great as pH 10 to 11 have been isolated from soils; they are mostly species of gram-positive Bacillus.

Text from:

“Bacteria and Other Monerans: Biosynthesis, nutrition, and growth” Britannica Online.

Applications

This knowledge can be applied through the use of the Rhizobium in places where the growth of legumes suffers from the soil. Since nitrogen is such an integral part of legume growth, Rhizobium must be present to transform the elemental nitrogen into a usable form. Often farmers will manually insert extra Rhizobium into their cropland to aid in their development. The ability to culture Super Rhizobium which can endure harsher environments such as an extremely acidic or basic soil, would allow agriculturists to grow plants in formerly unusable soil after a reclamation and the bacteria was added.

In the laboratory, the altered Rhizobium could be used in experiments with plants that a side effect of what was being tested caused a change in the pH of the nutrient solution.

Recommendations

For future research in this subject, it would be interesting to experiment with the pH levels. For instance higher and lower combinations might produce a wide range of results. One major suggestion would be to lower the basic side of the spectrum. The results from the switched spectrophotometer results show that a lower basic level would be better to start off with and then to culture that bacterium to a larger resistance.

The usage of other chemicals to alter the pH of something is also something worth looking into for future work. Instead of the usage of buffer solutions to raise or lower chemicals such as hydrochloric acid.

Conclusion

The testing done in the research turned out to be somewhat interesting as far as statistical information. The overall picture shows that the bacteria was affected by altering its environmental pH level.

The disk diffusion test, in which I measured the depression of growth around the disks (also called Halo s), the control was unaffected for the most part. The pH 4-disk applications showed significant results of having an effect. The mean size halo for the pH 4 grouping turned out to be 7.75 mm while the pH 10 grouping was lower at 4.50. Statistics show with a 99% confidentiality rate that the groups are significantly different. All standard errors do not overlap. The pH 4 Disks seemed to weaken that sample of bacteria.

The next test, a spectrophotometer transmittance test, in which scrapings from the most well defined halo s of both petri dishes were used to inoculate three neutral broths. The broths were allowed to grow and then a sample of each bacteria culture was measured. The results of this test were unexpected compared to the previous disk diffusion results. The less percent transmittance shows a larger amount of growth. The actual results are completely opposite of the suggested model of the disk diffusion results. The Control group had the highest percent transmittance, the pH 10 group was second highest, and the pH 4 scoring the lowest. These results show that the pH 4 group grew the most. The only theory I can offer for this is that the initial shock of the pH 4 solution in the DD(disk diffusion) test increased the strength of the bacteria allowing it to reproduce quicker. The weakness of the control and the pH 10 group can possibly be attributed to the converse of this.

The second spectrophotometer test also depicts an interesting twist. In this test the bacteria from the three broths were used to inoculate another three broths, which had their pH level altered. The bacteria from the pH 4 broth was placed into the new pH 10 broth, and vice versa for the old pH 10 broth. The control stayed the same, and was just inoculated into a new broth with the same pH of 7. The results show that the old pH 4 bacteria was killed by the environment of the pH 10 broth. The old pH 10 bacteria did well in the new pH 4 broth, and the control improved over the last test.

The effects of the changes in the pH are well defined. Further research would be required to pinpoint the weaknesses and strengths that are gained by sub-culturing. A breakdown of the individual tests into smaller tests and a wider spectrum of pH levels would, in my opinion, reveal more of the mystery.

Statistical Explanations

The mean of each of the groups is a cumulative average of the individual test data entries. The means under each group represent that group alone, and the statistics mean is the mean between the groups. The SE, or Standard Error, describes the chance of a possible data entry being within that range. This protects against Fluke data. Using the standard error you can tell if samples are Statistically different if the standard error bars (vertical lines on the bar graphs) do not overlap. The df, or the degrees of freedom, describe the sample size of the test and are an attribute to the power of the test. The Probability, or Prob., which is on the statistics sheet, corresponds to the chance of the samples being the same. An extremely low value means it is extremely unlikely they are the same. On the converse of that, the Confidence level is the percent chance the samples are different. A 95% or higher confidence level means there is a statistical difference between the samples.

The statistics for this test show that the groups are statistically different. The confidence level is 99%.

The statistics for this test also show that the groups are statistically different. The confidence level is 99%.

The statistics for this test again show that the groups are statistically different. The confidence level is 99%.

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