Kinetics Rate Law of Lactobacillus

Paracaseis Nitrogen Reductases

Hibah Ullah and Tu Vo
NEWMAN UNIVERSITY PHYSICAL CHEMISTRY

Abstract
Lactobacillus paracasei, a gram-positive, facultative fermenter, is a useful bacteria in the
study of kinetics. One useful aspect of this bacteria is the observation of the reduction of nitrate
into nitrite under anaerobic conditions. Lactobacillus bacteria was obtained and grown in an
MRS broth. Using a nitrate ion-selective electrode and the Logger Pro 3 software, the kinetic rate
of the nitrate reductase was determined. Although these results were relatively inconclusive due
to experimental error, both the nitrate disappearance and appearance were observed, and a rate
law equation was deduced from the experimental data (see results).
Keywords: Lactobacillus Paracasei, nitrate ion-selective electrode, kinetics, rate law

Introduction
Lactobacillus paracasei is a gram-positive, rod-shaped, facultative fermentative species
of lactic acid bacteria. This bacteria is commonly found in various places in the the human body
including the mouth, intestinal tract, and vaginal canal. This bacteria is also commonly found in
live fermenting products, such as yogurt and other dairy products. This experiment made use of
the lactobacillus nitrate reduction abilities. Until about 1955, lactobacillus bacteria were
believed to be unable to reduce nitrates. However, the Costilow & Humpherys study (1955)
showed that 18 of the 38 known strains of Lactobacillus reduced nitrates. One of these strains
included the paracasei strain, which is further examined in this study. 1
As stated earlier, lactobacillus is a facultative anaerobic bacteria. This type of bacteria
can produce lactic acid as a by-product of glucose metabolism in aerobic conditions. However,
under anaerobic nitrate respiration, the bacteria reduces nitrate to nitrite. This experiment will be
observing the rate at which nitrate is reduced to nitrite by monitoring the disappearance of nitrate
from solution. This monitoring is conducted by a Vernier Nitrate Ion-Selective Electrode. This
ion electrode is a sensor, or transducer, which converts the activity of a specific ion dissolved in a
solution into an electric potential; using this type of electrode offers great ion specificitythat is,
there is rarely any interference of other molecules with the electrode, allowing for a clear nitrate
reading to occur. 2 This electric potential made by the electrode is then read by a computer
program, in this case Logger Pro 3. This program allows for precise data analysis, as shown in
the results section of this document.

Procedure
The MRS broth was made using Sigma Aldrichs MRS powder (ingredients listed in table
#1) Approximately 2.08g of the MRS powder was weighed and added to a 500mL volumetric
flask containing 100mL of distilled water. The flask was gently shaken and then an additional
300mL of distilled water was added to the flask. A stir rod was dropped into the flask and the
mixture was heated and stirred for approximately 25 minutes to ensure the complete dissolving
of the MRS powder.
After all of the powder was dissolved, foil was used to securely cover the flask, and
sterilization tape was placed onto the foil. The flask was then autoclaved at 121C for 25 minutes
and allowed to cool for an hour and a half.
A pure sample of Lactobacillus was obtained and cultured on MRS agar prior to
experimentation. Two sterilized 50-mL conical centrifuge tubes were obtained, and once the
MRS broth was completely cooled, approximately 40 mL of broth was poured into each tube. A
round tip metal loop was sterilized using a flame and acetone, and was plunged into the MRS
agar. The loop was then inserted into one of the centrifuge tubes and mixed into the MRS broth.
This plunging process was repeated three more times to ensure that bacteria had entered the
MRS broth. This inoculation process was repeated for the second centrifuge tube. The tubes were
incubated for 36 hours to allow for optimal growth conditions.
To make the nitrate solution, 0.3286 g of pure potassium nitrate was dissolved in 13mL of
the MRS broth stock. This nitrate solution had roughly the concentration of 0.25 M KNO3. To
create a dilutions of this solution, 4 mL of this nitrate solution was pipetted into a 150 mL beaker
containing 96mL of the MRS stock, resulting in a 0.01 M NO3 solution. The next dilution was

made using 8mL of the MRS with potassium nitrate stock solution into 92mL of the MRS stock,
which was also added into a separate 150mL beaker, resulting in a 0.02 M NO3 solution.
The lactobacillus tubes were retrieved from the incubator after the incubation period was
over. The tubes were inverted several times. Next, the cells were counted using a hemocytometer.
The glass slit and coverslip were thoroughly cleaned with alcohol and the coverslip was gently
moistened using distilled water. The coverslip was then affixed to the hemocytometer. The
lactobacillus bacteria in the tubes were shaken gently to evenly distribute the cells in the mixture.
Before the cells settled, 0.5mL of the cell suspension was retrieved using a micropipette and
dispensed into an Eppendorf tube. Approximately 100microliters of the cells were then
transferred into a new Eppendorf tube and 400microliters of Trypan Blue was added into the
tube. The tube was shaken generously and gently. Using a pipette, a small drop was applied to
the hemocytometer and the coverslip was placed on the sample. The microscope was then used
to count the cells in the gridlines.
To measure the rate at which nitrate is converted to nitrite by the bacteria, a Vernier
Nitrate Ion-Selective Electrode was used. Using the Logger Pro 3 software, nitrate standards (1
g/L and 100 g/L) were used to calibrate the instrument. Once calibration was completed and the
cells had been counted, 5mL of the bacteria solution was added to each of the beakers containing
the MRS broth and nitrate solution and the electrode was placed into the beaker with the lower
nitrate concentration and the recorder was started. The second beaker was placed into a
refrigerator to inhibit further growth of the bacteria. After the data of the initial run was
completed, the procedure was repeated for the second beaker. Results were recorded and
analyzed using the Logger Pro 3 software and are shown below.

Table #13
MRS Broth Ingredients
Peptone
Meat extract
Yeast extract
D(+)-Glucose
Dipotassium hydrogen phosphate
Sodium acetate trihydrate
Triammonium citrate
Magnesium sulfate heptahydrate
Manganous sulfate tetrahydrate

Grams/Litre
10.0
8.0
4.0
20.0
2.0
5.0
2.0
0.2
0.05

Results

Figure #1: [NO3-] of 0.01 M KNO3 using Logger Pro 3

Key:

[NO3- ] of standard MRS media: very, very small

[NO3- ] of +0.01 M KNO3: As shown above
[NO3- ] of +0.02 M KNO3: No data/erroneous data
Data Analysis
There were several errors throughout this lab experiment. Lactobacillus paracasei is a
gram-positive, facultative fermenter. Prior to beginning the experiment, however, the incorrect
assumption was made that lactobacillus bacteria reduce nitrates via its nitrate reductase
enzymatic activities, converting nitrates to acids, as shown below.
[NO3-] + Carbohydrates + nitrate reductase lactic acid + nitrites + nitrate reductase
What was observed, however, was an increase of nitrates. This was because the oxygen present
throughout the running of these tests; a major error that occurs, therefore, was the improper
maintenances of an anaerobic environment throughout the experiment. In an aerobic
environment, carbohydrates in the media were broken down by the bacteria, resulting in an
increase in nitrate concentration as seen in figure #1.
Figure #1 shows the two nitrate concentration runs that were conducted using the
lactobacillus. In the second sample, sufficient data was not obtained for unknown reasons. In the
first sample (as shown in Figure #1), the detector showed a decrease of nitrates in the first 250
seconds. This is because the flask containing the Lactobacillus was completely closed for safe
transfer to a separate setting. On the other hand, there is an increase of nitrates after the flask is
opened.
Hence, the second sample can provide both the kinetics rate which nitrate reductase
catalyzes the consumption of nitrate in an anaerobic environment and the generation of nitrate in
an aerobic environment. Below are key data points taken from Figure 1.

Time (s)

mg/L NO3
0
188.9
2475
180

29220

220.4

Using the data, 8.9 mg/L of nitrate is consumed within 2475 seconds and 40.4 mg/L of nitrate is
generated within 26745 seconds.
Converting mg/L to molarity, it is 0.00014355 M and 0.0006516 M, respectively. Assuming that
there is insignificant growth from the time adding the Lactobacillus to the media, there are 50
million cells throughout the process. Calculating the number of atoms which each cell consume
and generate nitrate, we have the formula:
M*N/50,000,000/t = R where M = molarity; N = avogadro's number; t= time in seconds; R=rate
Rate of Consumption Under Anaerobic fermentation
Thus, the rate of consumption of nitrate when Lactobacillus undergoes fermentation is:
R = (0.00014355*6.022*10^23)/(50000000 *2465)
R = 7.01 * 108 NO3 molecules per second per cell.
Rate of Generation Under Aerobic Metabolism
Thus, the rate of nitrate generation under aerobic environment is:
R = (0.0006516*6.022*10^23)/(50000000 * 2465)
R= 3.18 * 109 NO3 molecules per second per cell.

Discussion

Per the calculations, the estimated kinetics rate of lactobacillus under an aerobic system is a
positive 3.18 * 109 NO3 molecules per second per cell. Due to limited resources and time, only
one set of data is obtained. To calculate for the rate law which Lactobacillus, with its nitrate
reductases, catalyzes nitrates cannot be determined. To do so, a minimum of two data sets is
required. Likewise, the estimated kinetics rate of lactobacillus under an anaerobic system is a
negative 7.01 * 10^8 NO3 molecules per second per cell. Due to limited data, the rate law for
this scenario is also undeterminable.
Because the rate law cannot be determined based on limited data, much of the continuing
discussion will be based on its potential and what could be done if more data is collected. First
and foremost, the consideration of multiple and varying concentrations of nitrate reductases in
each cell may play a huge role in the rate law per cell. However, on the grand scheme of the
experimental system, the numbers calculated are an average per cell sufficient for the purposes
here. For further analysis, the experiment may expand to estimate the average number of
reductases per cell. However, as it stands, the nature of this experiments methods is designed to
calculate for the kinetics rate law per cell, where each cell is considered as a single catalytic
entity.
Hence, should the experiment be completed with multiple sets of data, the rate law can be
determined and expressed by the Michaelis-Menten equation:

Conclusions and Limitations

Lactobacillus paracasei, being an anaerobic bacteria, was possibly not the correct choice
of bacteria for this experiment. In order for this experiment to work for Lactobacillus, strict
anaerobic conditions must be applied (closed system for analysis). Another solution to this issue
is simply using a bacteria that reduces nitrate to nitrite in aerobic conditions, such as bacterial
strains from genera Pseudomonas, Aeromonas, and Moraxella. 4
In conclusion, the kinetic rate using the single data set from the nitrate ion-selective was
determined to be 3.18 * 109 NO3 molecules per second per cell. If the kinetic rate law is to be
determined, multiple trial must be run in the future in order to calculate the rate constant, k.

References
1. Rogosa, M. (1961). Experimental Conditions for Nitrate Reduction by Certain Strains of
the Genus Lactobacillus. Retrieved from http://citeseerx.ist.psu.edu/viewdoc/download?
doi=10.1.1.614.6355&rep=rep1&type=pdf
2. Southwest Center for Microsystems Education (SCME) (2011). Introduction to
Transducers, Sensors, and Actuators. Retrieved from
http://engtech.weebly.com/uploads/5/1/0/6/5106995/more_on_transducers_sensors_actua
tors.pdf
3. Sigma-Aldrich. 69966 MRS Broth. Retrieved from:
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/SigmaAldrich/Datasheet/1/69966dat.pdf
4. Carter, Spiro, Richardson (1995). Soil and Sediment Bacteria Capable of Aerobic Nitrate

Respiration. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC167561/