The Bomb Bio-Remediation Squad Wore Wool
by Guest Blogger,
Tyler Kokjohn, Ph. D.
Dr. Morrie Craig is a large person who really fills a room. I remember him as a usually smiling, remarkably fast-moving man on a mission. During most of my time with him at Argonne National Laboratory I just tried to keep up.
The Environmental Research (ER) Division was working to devise methods to clean up soil and water contaminated by munitions manufacturing activities. The Joliet Army Ammunition Plant began operations in 1940 (1, 2) and by the time we started our research project some of the waste materials had incubated on that site for almost a half century. These discards do not improve with age and by the time I saw the material known as ‘red water’ (3) it had weathered into a deep maroon color. The toxic leftovers of processes used to purify 2, 4, 6-trinitrotoluene, also known as TNT or dynamite, red water is an extremely persistent hazardous waste. We were seeking microorganisms that could degrade red water into less toxic compounds, a process dubbed bio-remediation. Let’s say our efforts were not yielding enormous breakthroughs. Dr. Craig said he had the answer.
Sheep. That’s correct, the answer to the red water bioremediation dilemma that no one in the ER Division could solve was sheep. More accurately, Morrie told us microbes inhabiting sheep rumens could degrade TNT quickly and might be the key to cleaning up toxic wastes that had been lying around munitions plants for decades.
Morrie and his collaborators at the Oregon State University College of Veterinary Medicine had studied sheep (ovine) ruminal bacteria for quite a while. Their interest was sparked by the problems posed by plants such as tansy ragwort that are sometimes eaten by grazing animals. Pyrrolizidine alkaloids present in tansy are lethal to cattle and horses consuming them (4). However, sheep eating tansy usually do not die or develop cirrhosis, leading to the hypothesis that their livers have detoxifying enzymes cattle and horses lacked. A number of experiments revealed that detoxifying enzymes were the key to tansy resistance. Unexpectedly, it was recognized that sheep livers did not contain the protective enzymes, they were in the bacteria inhabiting the sheep rumen. Sheep could eat tansy because their ruminal bacteria detoxify the pyrrolizidine alkaloids before they ever reach the liver. Morrie had evidence these bacteria would work against a range of nasty chemicals including TNT and immediately recognized how they might solve a difficult bioremediation problem.
It is hard to describe the level of disdain my colleagues and I had for Morrie’s unbelievable idea. However, that mattered not one bit to this force of nature and he accepted a challenge to travel from Corvallis, Oregon, to Argonne National Laboratory, located just outside of Chicago, to prove his outrageous method worked. But this demonstration was not a simple matter because the only sources of the necessary bacteria were sheep. So Morrie packed up two sheep and hit the road.
There were other issues as well. One was that Argonne National Laboratory was not equipped to board and care for sheep; Morrie had to find a nearby facility that could house them properly. Working with obligate anaerobe bacteria is challenging because any exposure to oxygen kills them immediately and special methods and equipment are necessary to culture them. Now imagine taking extremely perishable samples from the sheep that were miles away from the lab and getting them back where you could work on them. Morrie and his graduate student, Dan Wachenheim, worked furiously to scrounge, improvise and cobble together a working anaerobe lab and chemical analysis facility. And somehow they made it all work.
I saw it work with my own eyes; living microbial ecosystem samples from sheep rumen quickly transformed TNT just like Morrie promised. Through the years Morrie has continued his investigations of sheep rumen microbes and produced a number of important contributions to our understanding of this amazing ecosystem (4, 5).
Working with someone like Morrie Craig was a rare privilege. Energy, enthusiasm and amazing positivity in the face of hostility were great lessons for a junior scientist to emulate. It wasn’t long before I had an opportunity in the form of oil flies (6). During their larval stages, these unique little insects inhabit places like the La Brea tar pits (7), happily immersed in thick, black, petroleum goop. The fumes coming off this stuff almost make you faint when you collect and work with them. Oil fly larvae devour animals which become trapped in the pits and ingest considerable amounts of tar in the process. Do they have a protective microbial gut flora that detoxifies oil products? We found they do have an interesting microbial ecology.
Twenty-five years have passed since Dr. Morrie Craig showed up at Argonne National Laboratory with some sheep and strange ideas. Far ahead of his peers, scientists and physicians are now discovering concepts he pioneered decades ago. One of Dr. Craig’s early papers on sheep ruminal bacteria noted how antibacterial treatments may unfortunately eliminate their resistance to tansy poisoning (4). These observations remind us to become more aware of our microbial companions and understand what we must do to enable them to continue performing their essential functions (8). The bomb bio-remediation squad may not wear wool today, but Dr. Craig and his colleagues still have some things to teach us.
(1) U. S. Forest Service. Joliet Army Ammunition Plant http://www.fs.usda.gov/detail/midewin/learning/history-culture/?cid=stelprdb5155180
(2) U. S. EPA. 2008. Collaboration Leads to Early Cleanup Completion. Joliet Army Ammunition Plant. https://www.epa.gov/sites/production/files/documents/success_story_joliet_0.pdf
(4) D. E. Wachenheim et al. 1992. Effects of Antibacterial Agents on In Vitro Ovine Ruminal Biotransformation of the Hepatotoxic Pyrrolizidine Alkaloid Jacobine. Applied and Environmental Microbiology 58(8):2559-2564. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC195821/
(5) R. W. Li et al. 2014. Metagenomic Insights into the RDX-Degrading Potential of the Ovine Rumen Microbiome. PLoS One 9(11):e110505 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226467/
(6) D. R. Kadavy et al. 1999. Microbiology of the Oil Fly, Helaeomyia petrolei. Applied and Environmental Microbiology 65(4):1477-1482. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC91210/
(7) The La Brea Tar Pits and George C. Page Museum of La Brea Discoveries. http://www.tarpits.org/
(8) Welcome to the Anthropocene – World War You Too? https://jayvay.wordpress.com/2016/08/31/world-war-you-too/