Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Civil and Environmental Engineering

Major Professor

Sarina J. Ergas

Keywords

chloramphenicol, dynamic modeling, precursor RNA, reverse transcription, wastewater

Abstract

A simple anaerobic digestion model has been developed for a continuously-stirred tank reactor (CSTR), which links the specific biogas production rate to the food/microorganism ratio (F/M). The model treats the various microbial populations involved in the sequential biological processes involved in anaerobic digestion as a composite and links the entire biomass specific growth rate directly to the specific biogas production rate. The model was calibrated by determining the specific gas production rate for a range of F/M values using a municipal wastewater seed sludge. The model predictions for steady-state biogas production rates were compared to observed biogas production and volatile solids destruction results from three laboratory-scale anaerobic digesters that were operated at hydraulic retention times of 10, 15, and 20 days. The F/M model results were shown to agree with reactor biogas output for 10, 15, and 20 day hydraulic retention times to within 5.0%, 14.3%, and 9.5%, respectively. A commercial wastewater treatment plant model, BioWin 3, was also used to model anaerobic digestion as a comparison. Agreement for the BioWin 3 model results, as compared to the 10, 15, and 20-day hydraulic retention time reactors, was within 66.2%, 114.1%, and 105.1%, respectively. In all cases the BioWin 3 model over-predicted biogas output as compared to the reactors.

A molecular biology method called RT-RiboSyn was developed to measure the specific growth rate of microbial populations. RT-RiboSyn, is an ex situ method that utilizes a reverse transcription and primer extension (RT&PE) method to analyze the rRNA extracted from a time series of samples treated with chloramphenicol. The method measures the rate of ribosome synthesis over time through the increase in precursor 16S rRNA (pre16S rRNA) relative to the mature 16S rRNA (16S rRNA). A single fluorescently labeled primer that targets an interior region of both pre16S and 16S rRNA for a distinct population is used to generate two pools of reverse transcription product. The ratio of pre16S and 16S rRNA is then determined by separating these pools by length using capillary electrophoresis, and measuring the fluorescent intensity of each pool of fragments.

Results from three different log growth cultures of Acinetobacter calcoaceticus indicate that RT-RiboSyn, as compared to spectrophotometer readings, was able to predict specific growth rates within -3.1% to 10% and -3.3% to 21.0% when using a primer targeting Eubacteria and Acinetobacter, respectively. The RT-RiboSyn results from a stationary phase culture predicted no growth and possible 16S rRNA degradation.

Further work was completed to determine whether the RiboSyn method would successfully measure growth rates of specific microbial populations in environmental samples. The first of these was activated sludge from a high-purity oxygen system in a wastewater treatment facility located in Tampa, Florida. The organism targeted was the Acinetobacter genus, which was shown to be prevalent via fluorescence in situ hybridization results. RT-RiboSyn results indicated that growth was not measureable for the Acinetobacter present in the system; however, since the sludge was taken at the end of the process, Acinetobacter may have been in stationary phase when the samples were collected.

Attempts were made to apply the method to methanogens in both pure culture and anaerobic digester sludge samples. An analysis of samples of RNA from Methanosarcina barkeri indicated that the presence of 16S rRNA could be measured; however, capillary electrophoresis instrument limitations prevented the detection of pre16S rRNA fragments. Additional testing of anaerobic digester sludge for both bacterial and Archaeal population was successful for detecting 16S rRNA and possibly precursor 16S rRNA fragments of a variety of lengths. However, specific growth rates could not be determined for the Archaea present in these samples, either due to capillary electrophoresis limitations or very slow growth rates. The results show that the RT-RiboSyn method is applicable to pure cultures; however, a modification of the method is needed to overcome the limitations apparent in populations with low specific growth rates.

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