The following 2 research projects are being conducted under the direction of Dr. Charles Kulpa, Director, Center for Environmental Science & Technology and are funded by Chevron Research and Technology Corporation, Richmond, CA. Dr. Mark Schneegurt is the Research Assistant Professor conducting the research with Sophia Dore, graduate student and Tom Beaven, technician.

Utilization of Molecular Probing (PCR and RT-PCR) to Analyze Mixed Cultures in Activated Sludge

The microbiological analysis of pollution control systems such as activated sludge is often limited to indirect methods such as plating, MPN, dye staining or microscopy. These methods have many drawbacks not the least of which is time frame in which results are obtained due to the growth rate of the bacteria. The use of polymerase chain reaction (PCR) to identify the presence of a specific organism or gene in a mixed culture is an invaluable tool for studying mixed microbial populations in waste water treatment system due to it's specificity and the short time to obtain results. However, PCR does not provide information on the expression of the gene in question or the activity of the bacterium being detected. This information has typically come from biochemical and analytical data. We have been utilizing PCR with reverse transcriptase-PCR to provide information on the activity of specific genes in a pollution control systems coupled with PCR to give a real time view of the activities of microorganisms in activated sludge. This research has focused on phenol degradation and nitrification for the removal of ammonia. RT-PCR has successfully been applied to study the expression of the dmp N gene in a sequencing batch reactor degrading simulated waste waters from the refining of petroleum. A portion of this work was recently published (Selvaratnam, S., Schoedel,B., McFarland, B., Kulpa, C. 1995 Application of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for Monitoring Expression of the Catabolic dmp N Gene in a Phenol Degrading Sequencing Batch Reactor. Appl. Environ. Microbiol. 61:3981-3985(1995). A second publication is in preparation. This research has been extended to the process of ammonia removal involving Nitrosomonas and Nitrobacter, which results in the conversion of ammonia to nitrate. This process is used by the petroleum refining industry to lower the ammonia content of their waste waters. It is the only economical process available at present. In our study, the Nitrosomonas sp. are being monitored using the amo A gene which codes for ammonia monoxygenase. The Nitrobacter sp. are being monitored by means of a specific 16s rRNA probe. PCR and RT-PCR are being used to study the impact of various environmental factors on these cultures in continuous stirred tank reactors. These studies will provide critical information on the behavior of these organisms but also demonstrate the potential and application of PCR and RT-PCR as a means of analysis in waste water treatment systems for slow growing, difficult to culture microorganisms.

Application of PCR and RT-PCR for Monitoring Nitrosomonas spp. and Nitrobacter spp. in Nitrifying Bioreactors.

S. Selvaratnam, B. L. McFarland, and C. F. Kulpa.

Nitrification is a key step in the nitrogen cycle and is often used in bioreactors to reduce the ammonia content of wastewaters produced during the refining of petroleum. Biological nitrification takes place in two distinct steps: oxidation of ammonia to nitrite, followed by conversion of nitrite to nitrate, by the activities of two genera of aerobic, chemolithotrophic bacteria, Nitrosomonas and Nitrobacter, respectively. The slow growth rate of nitrifiers combined with the fact that they comprise only a nominal fraction of the indigenous bacterial population make it difficult to track them using such conventional methods as MPN, activity testing, and fluorescence immunoassay measurements. The objective of this study was to determine the activities of nitrifiers in a nitrifying bioreactor using molecular techniques such as PCR and RT-PCR. Samples of biomass from lab-scale, activated sludge reactors were lysed by freeze-thawing and target DNA was detected by PCR. For determining expression of target genes by RT-PCR, nucleic acids were first treated with DNase prior to RT-PCR amplification. Extraction of nucleic acids by freeze-thawing and amplification by PCR or RT-PCR resulted in the detection of amo DNA/RNA (ammonia monooxygenase; characteristic of Nitrosomonas spp.) and DNA/RNA from a 16s rRNA gene specific for Nitrobacter spp. The results indicated that in the absence of nitrification or when a decrease in ammonia or nitrite removal was observed in the reactors, a corresponding absence or decrease in gene expression was detected. The results of this study will significantly improve the monitoring of nitrification in wastewater treatment.