Graduate Thesis Or Dissertation


Ruminal Biodegradation of Heterocyclic Toxic Compounds Public Deposited

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  • Cyclic nitramines are a class of compounds that include most of the commonly used explosives today. These are among the most common toxicants released into the environment as a result of human activity, generated on military ranges, battlefields, and production sites. Of these, hexahydro-1,3,5-trinitro-1,3,5- triazine (RDX) is of particular interest, due to its high stability and utility. This compound carries renal, hepatic, and neurological effects, and is suspected of being a carcinogen. In terms of remediation, RDX is readily drawn up by plants, which will either suffer toxic effects or will concentrate the compound but be unable to degrade it. The cyclic nitramines bear chemical similarities to plant toxins, in particular the pyrrolizidine alkaloids (PAs). PAs are interesting from the standpoint that they are only toxic to some species of ruminants – cattle are severely affected, while sheep and goats are not. This difference comes down to ruminal bacteria populations – that is, the bacteria that populate the sheep and goat rumen are capable of detoxifying PAs, while those that populate cattle rumens are not. Considering the chemical similarities, then, it was proposed that a potential solution for RDX degradation from contaminated sites would be to use grasses to draw it up and to use sheep to consume the grass, using their ruminal microbiota to detoxify the compound. Initial trials of aspects of this methodology were successful; ruminal isolates were capable of degrading several common cyclic nitramines including trinitrotoluene (TNT) and RDX. This document, then, aims to add to the body of research on this combined system in several ways. First, to further study effects of common plant toxins on ruminants through use of gene expression profiling via spotted microarray; second, to determine shifts in bacterial populations in the rumen via high-throughput 16S amplicon sequencing in an attempt to isolate sources of activity; thirdly, to study the whole genome of an RDX degrader via whole genome sequencing. The effects of the endophytic alkaloid ergovaline were studied in a host of cattle. In this study, four cattle were provided feed containing ergovaline over an extended period and liver samples taken at the start and end of the trial. Samples had mRNA extracted, which was used in a dual hybridization experiment to spotted microarrays for the bovine transcriptome. Top results were validated with RT-PCR. This study found responses in genes associated with intracellular transport, apoptosis, and RNA transcription control. In the second study, shifts in the population of sheep rumens in response to RDX were examined via sampling sheep rumen fluid, introducing a spike of RDX to each, and extracting whole DNA at several time points in order to amplify 16S rRNA genes. 16S amplicons were sequenced and OTU counts were contrasted between experimental and control groups via use of a linear mixed model (LMM) with logit transformation. This study found that individual animal contributed more to observed populations than any other factor, necessitating the use of the LMM to remove it as a random effect. Once this effect was countered, a clear pattern emerged of relative population increases in the phylum Bacteroietes, and more specifically, within taxa of Paraprevotellaceae. Finally, to study a RDX degrader in detail, genomic DNA from Sporanaerobacter acetigenes was sequenced. This species was previously found to degrade RDX, and so it was thought that a draft genome would aid in understanding processes of RDX detoxification in the rumen. While the draft genome was completed, however, initial results indicating degradation could not be reproduced, leading to development and validation of a new extraction method for RDX from anaerobic bacterial growth media. In summary, this research examined the Phyto-Ruminal-Biodegradation system on multiple levels, from that of the animal to ruminal populations as a whole to an individual species.
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