Journal of Undergraduate Research
Volume 4, Issue 2 - October 2002

The Effect of Exposure to Oxygen on Diauxic Lag of Pseudomonas denitrificanse

Ian Watson

ABSTRACT

Experiments were performed to investigate the effect of oxygen exposure on the diauxic lag and specific growth rate of Pseudomonas denitrificans. The experiments involved three phases: a period of anoxic growth under limited substrate conditions, next, an aerobic period with either growth (substrate present) or oxygen exposure (no substrate), followed by a final period of anoxic growth. In the third phase of the experiment, the lag and specific growth rate of the biomass that underwent growth was compared to that of the sample that was only exposed to oxygen. Bacterial growth was monitored by measurement of the absorbance of biomass samples in a spectrophotometer.

INTRODUCTION

Malaria One of the most important problems facing any society is the management of the vast amounts of wastewater produced by its cities, industries and agriculture. Most civilized countries have adopted treatment programs that can cleanse and return wastewater to natural water sources, including rivers, lakes and aquifers, with a minimum amount of environmental harm via chemical and biological treatment methods.

Among the most important steps in water reclamation is the removal of harmful chemicals by activation of mixed bacteria cultures (activated sludge), which are already present in wastewater. This "activation" begins the bacterial digestion of the many chemical species present in the waste. Some contaminants present in untreated wastewater are phosphates, nitrates, and sulfates (Ramalho).

Pure culture characteristics of the bacteria present in activated sludge have been a large focus of recent research. These pure culture studies are necessary because the behavior of a single species of bacteria cannot be deduced from the experimental data acquired in the presence of other bacteria, as in activated sludge experiments. Knowledge of the pure culture growth characteristics of a bacteria species can be utilized in the development of a characteristic mathematical model for the behavior of that species. This model, in turn, can be used to better predict the behavior of activated sludge within a wastewater treatment plant (Liu). This paper details one such experiment, wherein some characteristics of the behavior of Pseudomonas denitrificans are investigated.

Nitrogen compounds are present in waste as ammonium, nitrite, and nitrate ions (Ramalho). The value of Pseudomonas denitrificans lies in its ability to effectively remove nitrates and nitrites from wastewater.

The biological water treatment process occurs in two phases--an aerobic phase (O2 dissolved in solution) followed by an anoxic phase (O2 stripped from solution). During the aerobic growth phase, nitrifying bacteria (nitrosomonas and nitrobacter microorganisms), oxidize ammonium to nitrite and then to nitrate (Ramalho):



During this aerobic phase, Pseudomonas denitrificans undergoes growth with oxygen as its electron receptor. Switching to an anoxic phase allows this bacterium (along with other denitrifying bacteria) to utilize nitrate and its reduction products (NO2-, NO, N2O) as an electron receptor, reducing nitrate and nitrite to harmless nitrogen gas, which is subsequently released to the atmosphere (Liu):




If these nitrogen compounds were not removed, the results would be far reaching. Ingested nitrogen compounds and their derivatives can cause several illnesses in humans and animals. Nitrates are responsible for the formation of algae blooms and growth of other undesirable green plants in still aquatic systems. Furthermore, if nitrates are not sufficiently removed, chlorination that occurs later in the water treatment process can lead to the formation of chloramines and nitrogen trichloride, both carcinogens (Ramalho).

In early studies with Pseudomonas denitrificans, it was noted that a growth delay occurs upon switching from an aerobic to an anoxic phase. This delay in growth between phases is called a diauxic lag (Monod). Furthermore, it was discovered that the amount of aerobic growth affects the length of the ensuing lag, and the growth rate of the subsequent anoxic phase (Liu). These effects result in a reduction in the quantity of nitrogen removed in the treatment process. A reduction in the length of the lag between phases could drastically increase the efficiency of the nitrogen removal process. For this reason, a great deal of work is being devoted to understanding the underlying mechanisms involved in the growth of this bacterium.

The mechanism under investigation in this experiment is the effect of exposure to oxygen on the lag of Pseudomonas denitrificans. More specifically investigated was whether the important factor affecting the length of the ensuing lag and anoxic growth rate is the length of aeration or the length of aerobic growth.

MATERIALS AND METHODS

An experiment begins with the revival of a freeze-dried culture Pseudomonas denitrificans (ATCC 13867) in 125mL of sterilized Nutrient Broth (Difco #0003-17-8) with shaking for two days. This broth is then transferred onto plates of sterilized tryptic soy agar under an ethanol-rubbed laminar flow hood, and allowed to incubate for two days at 35ºC. These plate cultures can then be stored with refrigeration at 4ºC for up to two weeks, or can be used immediately in an experimental run.

To begin an experimental run, simple nutrient solutions are prepared. This experiment requires three types of solutions, detailed in Table 1.

Table 1
A detailed list of components and their concentrations in the three types of nutrient solutions prepared for each experiment. All solutions are autoclaved and have pH adjusted to ~7.0 with 2M NaOH
Component Solution w/ No Substrate Solution w/ Limited Substrate Solution w/ Excess Substrate
Substrate
Glutamic Acid 0 g/L .131 g/L 5 g/L
Inorganics
K2HPO4 5 g/L 5 g/L 5 g/L
KH2PO4 1.5 g/L 1.5 g/L 1.5 g/L
NaCl 1 g/L 1 g/L 1 g/L
NH4Cl 1 g/L 1 g/L 1 g/L
MgSO4 / H2O 0.2 g/L 0.2 g/L 0.2 g/L
CaCl2 / H2O 0.0264 g/L 0.0264 g/L 0.0264 g/L
Nitrates
NO3N 4 g/L 4 g/L 4 g/L
Trace Metals (.5% w/V)
CuSO4 1 Drop/L 1 Drop/L 1 Drop/L
FeCl3 1 Drop/L 1 Drop/L 1 Drop/L
MnCl2 1 Drop/L 1 Drop/L 1 Drop/L
Na2MoO42H2O 1 Drop/L 1 Drop/L 1 Drop/L

Each run is performed in three stages. In the first stage, Pseudomonas denitrificans is inoculated directly from the agar plates to a 500mL Erlenmeyer flask containing the carbon-limited nutrient solution and stirred to homogeneity. Meanwhile, the remaining carbon-limited nutrient solution is added to a 2L continuously stirred bioreactor, warmed to 30ºC, and sparged with nitrogen gas. Sparging with N2 is necessary to strip the solution of all dissolved oxygen, creating anoxic conditions. When both the reactor and the inoculated solution have been prepared, the spectrophotometer (set to 550 nm) is zeroed with nutrient solution. The inoculated solution is then added in intervals to the reactor until the absorbance of the reactor solution is between 0.03 and 0.05. The bioreactor is sampled every 15 minutes and the absorbance noted.

It is important to note that this stage of the experiment acts as an in situ preculture, with the purpose of eliminating variability in further stages of the experiment (where the culture is divided between two reactors) by drawing on only one large preculture. In the first stage of growth the amount of carbon substrate present is chosen by calculation to limit growth of the culture to an absorbance between 0.08 and 0.12. The purpose of this technique is to ensure the total depletion of carbon substrate in solution before proceeding to the next phase of the experiment.

When growth in the first phase is complete, the culture is then divided between two reactors. Next, one reactor is charged with nutrient solution containing no carbon substrate, and the other with the same solution containing an excess of carbon substrate. Thee nutrient solutions are added to bring the reactors' absorbances to the same value (again between .03 and .05). The reactors are again brought to 30ºC, but are instead sparged with air in order to maintain oxic conditions. The absorbance of solution in each reactor is taken every 15 minutes over a predetermined length of time, ranging in these experiments from 1 hour to about 9 hours.

After the second phase of the experiment is complete, both reactors are again diluted to their initial absorbances with the nutrient solution containing excess carbon substrate. The absorbances are again measured every 15 minutes until both solutions have grown to about 2000% of their initial absorbance.

Figure 1 gives a visual summary of the experimental procedure detailed above:

Figure 1. Flow Chart detailing the experimental procedure.


Figure 1. Flow Chart detailing the experimental procedure.

ANALYTIC METHODS

It is important to note for this experiment that there is direct proportionality between the amount of biomass in a culture and the absorbance of that culture, which is given by the equation:

A = αX (4), where

α ≡ Constant
A ≡ Absorbance of a sample
X ≡ Biomass concentration

This proportionality allows for the direct use of the absorbance in experimental results.

RESULTS

In all, seven experimental runs were performed. The results of a single run are depicted in Figure 2. The three experimental phases are clearly demarcated abrupt drops in absorbance. A comparison of the two plots in the third phase of the experiment shows that the culture that did not grow aerobically began anoxic growth very quickly, while the other lagged for about 5.5 hours.

Figure 2. Absorbance versus Time. Vertical lines depict divisions between experimental phases.Figure 2. Absorbance versus Time. Vertical lines depict divisions between experimental phases.


Figure 2. Absorbance versus Time. Vertical lines depict divisions between experimental phases.

Because the growth of the bacteria culture is exponential in nature, a semi log plot can be utilized to help extract trends from the absorbance data. Figure 3 depicts the results of the same run in a semi log plot. The slopes of the trend lines shown are the specific growth rates of the biomass over those time periods. Note that the period with lag appears as a flat line (specific growth rate = 0).

 

Figure 3. ln (biomass) versus time

 

Figure 3. ln (biomass) versus time. Slopes of trend lines yield specific growth rates.

CONCLUSIONS

Through analyzing the data of each of the experiments in a semi log plot, both the length of lags and anoxic growth rates for the experiments were obtained. Overall, the experiments show that there is no difference in specific growth rate between cultures that grew aerobically and those that were merely exposed to oxygen. This result is in disagreement with earlier studies that were performed on activated sludge, where there was a significant reduction in specific growth rate in the culture that underwent aerobic growth (Oh).

However, the experiments did show a significant difference in the lengths of the lags under the two conditions. Diauxic lag occurred in all experiments after growth with oxygen, while very little lag (or no lag) was apparent in the culture that did not grow with oxygen. This effect is apparent in Figure 3. The experiments showed little or no correlation between the lengths of aerobic growth and the length of ensuing lags.

REFERENCES

Ramalho, R.S., Introduction to Wastewater Treatment Processes. Academic Press, Inc., San Diego, California, 1983.

Liu, P.H., Zhan, G., Svoronos, S.A., and Koopman, B., "Diauxic Lag from Changing Electron Acceptors in activated Sludge Treatment," Water Research, 32, 3452-3460 (1998).

Liu, P.H., Zhan, G., Svoronos, S.A., and Koopman, B., "Experimental and Modeling Study of Diauxic Lag of Pseudomonas Denitrificans Switching from Oxic to Anoxic Conditions," Biotechnology & Bioengineering, 60, 649-655 (1998).

Monod, J., "The Growth of Bacterial Cultures," Annual Review of Microbiology, 3, 371-394 (1949).

Oh, J., Silverstein, J. "Effect of Air On-Off Cycles on Activated-Sludge Denitrification," Water Environment Research, 71, 1276-1282 (1999).

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