Bacterial Metabolism and Enzymatic Growth

Jenny Nguy #20352562

BIOL 140L: Bacterial Metabolism and Enzymatic Growth

Introduction

Bacteria differ in the substances from which they derive energy and the enzymes that they produce. Enzymes are biological catalysts; they are substances produced by the body to control the rate of reactions within the body. Particular enzymes, exoenzymes, are produced in large quantities and are excreted into the cell’s environment where they speed up the catabolism of insoluble polymers for nutrients (Madigan, 2009). Endoenzymes, unlike exoenzymes are produced in small quantities and remain within the cell (Kocholaty et al, 1938).

The purpose of these experiments is to determine the enzymatic and metabolic activities of a variety of bacteria in varying environments. The environments in which the bacteria are observed contain the presence of indole, urease, hydrogen sulfide, and carbohydrates lactose, sucrose, and glucose. The species of tester bacteria cultures for metabolic activities are Escherichia coli (Ec), Pseudomonas aeruoginosa (Pa), Bacillus subtilis (Bs), and Proteus vulgaris (Pv). The enzymatic activities of bacteria are observed in the presence of the enzymes amylase, lipase, and protease. The bacteria used are Escherichia coli, Pseudomonas aeruoginosa, and Bacillus subtilis.

The metabolism of the aforementioned carbohydrates results in a change of acidity, evident by a colour change in the presence of the bromocresol purple indicator. Each carbohydrate broth is inoculated with two loopfuls of each bacterium. If the broth undergoes metabolism and becomes yellow in colour, it is acidic. The broth is basic if, after metabolism, it becomes dark blue in colour. The broth will remain purple if it is neutral after metabolism. The metabolism of particular carbohydrates by some bacteria may be accompanied by gas formation which will be collected in an inverted tube (Madigan et al, 2009).

Indole is a compound derived from the amino acid tryptophan. A broth of tryptone is inoculated with two loopfuls of each bacterium. If the bacterium is one which converts tryptophan to indole, a red surface layer appears upon addition of dimethyl-aminobenzaldehyde, more commonly known as Kovac’s reagent. Otherwise, the colour of the broth will remain completely yellow (Madigan et al, 2009).

In the urease test, a urea broth, yellow before incubation, is inoculated with two loopfuls of each bacterium. If the broth, after inoculation, increases in acidity, the colour changes to pink with the presence of a phenol red indicator. If no colour change has occurred, the urea has not been broken down, meaning that the bacterium does not produce urease (Madigan et al, 2009).

Bacteria that produce hydrogen sulfide are found in the intestines of animals. They are known as “enteric” bacteria – bacteria that are gram-negative, facultative anaerobes with a rod-shaped morphology. To test for the formation of hydrogen sulfide, a straight inoculation is made with an inoculating needle into a SIM agar butt. If hydrogen sulfide is produced by the bacteria, the SIM medium will blacken. If hydrogen sulfide is not produced by the bacterium, the SIM medium may become paler, as some motion of the bacteria may have occurred, however the colour of the medium will remain a pale yellow (Madigan et al, 2009).

Enzymatic activity on different agars is observed by the physical properties of and around the bacterial colonies. The three species of bacteria, previously mentioned, were spotted onto three different agar mediums, starch, tween 80, and milk and incubated for 48-72 hours at 37°C. If, on the starch agar, there was a clearing around the bacterial colony, the bacterium is one which hydrolyzes starch. To better see the clearing, Gram’s iodine solution was added to the agar plate and any starch present was stained. Lipolysis activity, which is the breakdown of fat, is evident if the area around which the bacterium was spotted on the tween 80 agar is cloudy. Proteolysis, the breakdown of protein, like starch hydrolysis, results in a clearing surrounding the bacterial colony. To determine the bacterium’s production of catalase, hydrogen peroxide was added to each colony. Catalase, if present, will result in the formation of oxygen bubbles. The lack of oxygen bubbles indicates a lack of catalase. The presence of oxidase was determined by the combination of Kovac’s reagent, which acts as an artificial electron acceptor, and undergoes oxidation by cytochrome c. A positive reaction is one in which the bacteria darken. A colour change will not occur if oxidase is not present.

The expected results for each bacterium in each medium can be found in table 1 and table 2 in the results section.

The ability to identify bacteria allows the ability to confirm the infection of such bacteria and distinguish between two similar bacteria. For example, salmonella and edwardsiella are similar in morphology and react similarly in various tests. The two bacteria may be differentiated with an indole test; edwardsiella produces indole and tests positive whereas salmonella does not.

Materials and Procedure

The materials involved, and the procedure by which these experiments were performed, are as stated in the experiments ...

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The expected results for each bacterium in each medium can be found in table 1 and table 2 in the results section.

Materials and Procedure

The materials involved, and the procedure by which these experiments were performed, are as stated in the experiments 18 and 19, outlined in the University of Waterloo Fall 2009 Biol 140L Lab Manual, pp69-77, titled Some Metabolic Activities of Bacteria and Bacterial Enzymes. No changes were made to the outline procedure.

Department of Biology 2009 Fundamentals of Microbiology Lab Manual. University of Waterloo, Waterloo. pp22-30.

Results

Table 1: Expected results for metabolic activity

Below are the expected results for carbohydrate fermentation, the conversion of tryptophan to indole, the production of urease, and the production of hydrogen sulfide in a SIM media by four species of bacteria: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Proteus vulgaris.

Legend (applicable to all tables):

A = acid production

K = alkaline acid production

N = neutral pH

G = formation of gas

+ = positive result

- = negative result

Table 2: Expected results for enzymatic activity

Below are the expected results for the hydrolysis of starch, lipolysis, proteolysis, catalysis and oxidation by three species of bacteria: Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa.

Table 3: Carbohydrate Fermentation

Below are the results obtained from the inoculation of E. coli, P. aeruginosa, B. subtilis, and P. vulgaris into broths of carbohydrates glucose, lactose, and sucrose. Metabolic activity is determined by the change in acidity, which is indicated by a change in colour. The indicator phenol red was added to the carbohydrate broths. Gas formation is also noted.

Table 4: Indole Production

Below are results obtained from the inoculation of E. coli, P. aeruginosa, B. subtilis, and P. vulgaris into broths of tryptone. Indole will be present if the bacteria is capable of converting the amino acid tryptophan in the broth to indole. Kovac’s reagent is added to determine if indole is present. If present, a layer of red will form at the surface of the broth.

Table 5: Urease Activity

Below are results obtained from the inoculation of E. coli, P. aeruginosa, B. subtilis, and P. vulgaris into broths of urea. If urease is produced by the bacteria, urea will catabolise into ammonia, which will alter the pH of the broth. Phenol red, a pH indicator present in the broth will change the colour of the broth if there is a change in pH.

Table 6: Production of Hydrogen Sulfide

Below are results obtained from the inoculation of of E. coli, P. aeruginosa, B. subtilis, and P. vulgaris into SIM agar butts. Ferric iron is present in the SIM medium and will react with the production of hydrogen sulfide, if produced by the bacteria. The result is a blackening of the medium.

Table 7: Exoenzyme Activity

Below are results obtained from the spotting of Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa onto agar plates of starch, lipids present in tween 80 and proteins, specifically casein which is present in the milk agar. The production of corresponding enzymes to the substances in the agar will alter the area surrounding the bacteria. The enzymes involves are amylase, lipase, and protease, respectively.

Table 8: Endoenzyme Activity

Below are results for the presence of catalase is obtained from the addition of hydrogen peroxide to the bacteria Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa on the tween 80 agar plate. The results for the presence of oxidase is obtained from the spreading of each of the aforementioned bacteria onto a piece of filter paper which had been wet with tetramethyl-p-phenylenediamine reagent.

Discussion

The results showed that the fermentation of E. coli and P. vulgaris in glucose increased the acidity and formed gas. However, the acidity of P. aeroginosa and B. subtilis remained neutral and no gas production was observed. The experimental results for Ec and Pv concurred with the expected results, however, Pa and Bs did not. The inoculation of Pa, Bs, and Pv into a lactose medium resulted in a neutral pH and no gas formation. Ec in a lactose medium results in the production of acid and gas. These results may be confirmed by the expected results except for Pa. All organisms when inoculated into a sucrose medium showed no colour change and no gas formation. While these results were expected for Ec and Pv, Pa was expected to indicate an alkaline change in acidity and Bs was expected to indicate an increase in pH. Pa, in lactose and sucrose mediums was not exposed to the medium for an efficient amount of time and did not change colour to indicate an alkaline change in acidity as expected. A difference in the experimental results from the expected results may be explained by a number of causes. Human error may have occurred and an insufficient amount of the bacteria may have been inoculated into the media. This would explain the lack of colour change in situations where a change should have occurred, such as with B. subtilis.

Fermentation is the process of catabolism of substances to derive energy for, in these experiments, aerobic bacteria. The final product of carbohydrate fermentation is short, branched fatty acids (Cummins & Englyst). The majority of carbohydrate fermentations, then, should result in an increase in acidity. Gas production is the release of hydrogen or carbon dioxide gase (Madigan et al, 2009). So, a change in acidity of the broth after inoculation by the organisms indicates that such media were fermented by the bacteria. If the pH of the broth remained neutral, the carbohydrate was not fermented.

Indole, a derivative of the amino acid tryptophan, is produced when tryptophan is metabolised. When dimethyal-aminobenzaldehyde is added to indole, a layer of red appears. Of the bacteria used in this experiment, only E. coli produced indole (Madigan et al, 2009). Pa, Bs, and Pv did not form a red layer when the reagent was added, and as such, are unable to metabolise tryptophan. These experimental results obtained agree with the expected results.

Urea is a substance which catabolises into ammonia and carbon dioxide by the enzyme urease. Urease is an endoenzyme produced by some bacteria. Of the organisms which were tested, only P. vulgaris produced urease and was able to break down the urea. The presence of ammonia when urea was broken down is responsible for the increase in acidity, which was indicated by the pink colour change of the broth. Urea is utilized by the bacteria as a source of nitrogen (Christensen, 1946). Ec, Pa, and Bs do not produce urease. These findings are in agreement the expected results.

The SIM tubes, in which the bacteria are inoculated with an inoculating needle, are used to determine the production of hydrogen sulfide. The results obtained shows that only P. vulgaris produces hydrogen sulfide, where as Ec, Pa, and Bs do not. This is confirmed by the expected results. The SIM medium contains ferric iron which undergoes single displacement with the hydrogen sulfide, if present, to produce iron sulfide. Iron sulfide is a black precipitate, and as such, would results in the blackening of the SIM medium. Hydrogen sulfide is produced when the amino acid sulfur is catabolised by bacteria (Madigan et al, 2009).

The starch agar was only hydrolyzed by B. subtilis, and indicated by the clearing which surrounded the bacterial colony. The clearing was not observed in E. coli or P. aeruginosa. These results are supported by the expected results. Starch is a compound composed of polymers amylose and amylopectin which are connected by 1-4 diglycan linkages. Normally, amylose retains iodine in its helices. However, when starch is broken down by amylase the area surrounding the bacterial colony can no longer retain the iodine (Kotarski et al, 1992). When Gram’s iodine is added to the agar, the area surrounding the Bs colony was not stained – Bs tested positive for amylase. Ec and Pa were dyed, indicating a negative result; amylase was not produced by these enzymes since the starch did not catabolise.

Lipolysis is the catabolism is lipids by the exoenzyme lipase. Lipids are triglycerides and are broken down to free fatty acids (Goldberg, 1996). It was present in P. aeruginosa, as indicated by the cloudiness surrounding the colony, however was no present in Bs or Ec. The results of Bs did not concur with the expected results. Bs was expected to produce lipase, but was not observed in the experimental results. This can be explained by a faint cloudiness that was not observed, although it may have been present.

The enzymes Bs and Pa tested positive for proteolysis, the breakdown of the protein casein, of the milk agar. The breakdown is caused by the presence of the exoenzyme protease. It is indicated by a clearing that surrounds the bacterial colony, termed the proteolytic zone. E. coli tested negative, as it lacked the proteolytic zone around the bacteria colony. These results match the expected results.

The presence of endoenzymes catalase and oxidase in the bacteria were tested. The results showed that all three bacteria, Ec, Bs, and Pa produced catalase. This was determined by the formation of bubbles when hydrogen peroxide was added. The hydrogen peroxide was catabolised and oxygen gas was released in the form of bubbles (Madigan et al, 2009). These expected results concur with the experimental results. The experiment which tested for the presence of oxidase was positive for Ec and Pa and negative for Bs. While the results for Bs and Pa were as expected, the test for Ec resulted in a false positive. Only a small portion of the test area changed colour, compared to Pa. The colour change may have been due to contamination of the filter paper with another substance. The colour change occurs as a result of the oxidation of Kovac’s reagent, which acts as a false electron acceptor, by cytochrome c (Madigan et al, 2009).

There are many potential errors which may have occurred to produce results that differed from the expected results. Contamination may have occurred, and was likely seen in the oxidation reaction of E. coli. An insufficient amount of bacteria may have been inoculated into the broths producing a very weak outcome which may not have been noticed. Human error may have occurred, such as in the observation of the results. For instance, the presence of a clearing as a result of exoenzyme tests may not have been seen, despite being present.

These tests are useful as they are simple tests which can be used to determine the species of a bacterium. This would be helpful in the diagnosis of bacterial diseases as it is an efficient way to obtain results at a low cost. For example, E. coli can be identified by an indole test. The disadvantage to these tests, however, is that an indole test alone may not necessarily mean that the bacterium is E. coli. It could also mean that the bacterium being tested also converts tryptophan to indole. Several tests would have to be done to confirm that the bacterium is E. coli, and doesn’t only have similar results.

In general, the experiments performed were successful. The majority of the enzymatic and metabolic activities of E. coli, P. aeruginosa, B. subtilis, and P. vulgaris were correctly observed. Most of the results obtained were those of the ideal. Most of the results obtained that were not those of the ideal results could be explained by a minor error which could be avoided if the experiment were performed again.

References

Christensen, W.B. (1946). Urea decomposition as a means of differentiating proteus and paracolon cultures from each other and from salmonella and shigella types. [Electronic Version] Journal of Bacteriology. 52, 461-466.

Cummins, J.H., Englyst, H.N. (1987). Fermentation in the human large intesting and the available substrates. [Electronic Version] American Society for Clinial Nutrition. 45, 1243-1255.

Department of Biology 2009 Fundamentals of Microbiology Lab Manual. University of Waterloo, Waterloo. pp22-30.

Goldberg, I.J. (1996). Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. [Electronic Version] Journal of Lipid Research. 37, 693-702.

Madigan, M.T., Martinko, J.M.,Dunlap, P.V., Clark, D.P. (2009). Brock Biology of Microorganisms, 12th Ed. Pearson Education Inc. New Jersey pp 747, 908.

Kocholaty, W., Smith, L., Weil, L. (1938). CCXIX. Students on the endo-enzymes, particularly the peptidases, of Clostridium Histolyticum. [Electronic Version] The Biochemical Research Foundation of the Franklin Institute. pp 1691 – 1692.

Kotarski, S.F., Waniska, R.D., Thurn, K.K. (1992). Starch Hydrolysis by the Ruminal Microflora. [Electronic Version] The Journal of Nutirition. 122, 178-187.