Friday, November 15, 2019

Tracking Chicken Sh*$ in Chesapeake Bay

Rapid Response Chicken Sh*% team
          The Chesapeake Bay has absorbed the Eastern seaboard’s agricultural and urban runoff for centuries ever since Europeans settled there in the 1600s. The chemical wastes took their toll in the 1960s with anoxic dead zones and toxic algal blooms. Laws were passed in the states within its watershed to eliminate phosphates in detergents and to watch out for rampant pollution from under-performing sewage treatment plants. The Bay was considerably cleaned up in the decades following but not before the oyster, blue crab and sea bass fisheries were almost destroyed.
            In the summer of 1998, a bloom of the noxious alga, Pfiesteria, caused massive fish kills. Pfiesteria, first described in 1988 by JoAnn Burkholder of North Carolina State University, is a dinoflagellate, an organism with a complex life cycle part of which is a stage that produces neurotoxins thought to be responsible for fish kills. In several locations in the Bay where the fish kills occurred, it was thought that a relatively new source of pollution was potentially impacting water quality. Pollution from the more than 6,000 unregulated chicken barns, each with 25,000 chickens, was indicated as the culprit. Stimulated by slugs of nutrients from chicken barns on the Eastern Shore, Pfiesteria was also thought to cause human health issues, but at the time this finding was controversial. Environmentalists stormed the state legislatures in Maryland, Delaware, and Virginia calling for regulation of chicken wastes. 
Typical Delaware chicken barn
            Chicken producers and farmers responded with claims that they were operating within the law. They demanded proof that the nutrients in chicken farm runoff were causing the deadly algal blooms in the Bay. Who actually owned the chicken waste (75,000 tons produced per year) was another matter that complicated things. Farmers, who built low-slung barns on their property to house the chickens, don’t actually own the chickens they are raising. The producer essentially loans the chickens to them to raise from hatchlings to pullet size birds. The farmers buy (and own) the chicken feed from the producer. No one wanted to take domain over the wastes.
One the farms we monitored
            Working with Mat Wooller on the biocomplexity of mangroves project, we discussed how and if isotope tracers might help in identifying whether the source of pollution causing the Pfiesteria blooms was chicken wastes. Chickens excrete a different form of nitrogen in their wastes that is chemically distinct from the nitrogenous wastes from cows, fertilizers, and human sewage pollutants. Their waste, uric acid, is a complicated breakdown product similar in complexity to the backbone structures of DNA and RNA. Wooller and I hypothesized that if we could trace uric acid from chicken farms into the ecosystem and into Chesapeake Bay waters, we’d know whether chicken waste was responsible for the blooms. In order for this to be conclusive, the uric acid would need to have a distinctive isotope signature that could be further traced back to chicken farming. 
Mat Wooller's cartoon of the flow of nitrogen
            I wrote a small proposal to the Smithsonian’s Mellon fund and received a fellowship to carry out the study. We started by learning to assay uric acid using a chemical test kit. Then, we purchased several hundred grams of isotopically labeled uric acid and designed an experiment to follow its concentration and its incorporation into the organisms within a small watershed on Smithsonian’s Environmental Research Center. The isotopically labeled uric acid was expensive, a few thousand dollars. We mixed it into a large vat of stream water, adjusted the concentration, and had a special pump drip the solution into a 1-meter wide creek. We’d designed a week-long experiment and were monitoring it every 12 hours by collecting creek water over its 50 meter reach as the stream emptied into the Bay.
            During the second day, our uric acid tracer disappeared. We were puzzled and checked our assay method. It worked. We scratched our head. The next morning we took a sample from the vat of uric acid solution and found that all of the uric acid was gone! Native microbes had converted it quantitatively to ammonium. Our experiment was a failure or so we thought. We learned two things: uric acid, whether from a bottle or from the rump of chicken, will convert swiftly to ammonium, and elevated levels of ammonium can be detected in streams and enter the food web.
            With summer intern Quinn Roberts, we developed a field plan for taking samples at the edges of chicken farms that had large manure piles on their property. Ditches that drained the land surround most chicken farms on the Eastern Shore of Maryland. These farms also had the majority of their land under some sort of crop, typically corn or soybeans. We had no interest in investigating individual farmers, who were growing chickens and earned very little for their efforts. Ultimately the big producers like Perdue or Holly Farms were making substantial profits. We were careful to obtain our samples without being detected by the farmers though. We collected a few leaves from crops plants, a baggie full of soil they were growing in, a water sample from any standing water, and if at all possible, a handful of chicken waste. Chicken “waste” or manure is actually a combination of chicken excrement, straw from the birds’ bedding, and bits of feathers. Scooping up the chicken waste was the most daring thing we did—often casing the “job”, swinging the car around, then streaking out with gloves on, grabbing a hasty sample, before racing back to the car and pealing away. One of intern Allie Gales' favorite stories is having her run to a manure pile with gloves. When she said: "I'm nervous!" I said: "Just tell them it's for your grandma's petunias if anyone asks!"
            Occasionally locals asked us what we were doing in their neighborhood. I dressed in plaid shorts, a pink polo shirt, and looked nothing like a senior scientist. I told curious folks that we were lost and looking for the way to the beach. Mat in baggy cargo shorts used his thickest British accent and claimed he was a British Botanist touring America. Quinn Roberts wore a college T-shirt and short gym shorts. Her cover was as a student who needed to have a research project to graduate. Near the end of our study, we still had not sampled an actual chicken. Quinn bravely knocked on a farmer’s door and asked for a sample. She was taken to one of the barns and handed a scrawny nearly-dead chicken, which Quinn had to dispatch. For 6 months afterwards, she was a vegetarian.
Mat and Quinn monitoring a farm drainage ditch
            We found uric acid, the chicken’s waste compound, only once in mid-December when it was cold and microbial activity was slowed such that we could actually catch the uric acid before it was converted to ammonium. Meanwhile, we were measuring all of the nitrogen compounds (i.e., ammonium, nitrate, nitrite, and organic nitrogen) in the samples. We could tell based on the concentrations and the isotope compositions that the nitrogen had originated from chicken manure. The isotope signatures showed an enrichment of the heavy isotope of nitrogen (15N) that could be traced from the chicken waste to the soil to the crops and into streamside vegetation. As in many ecosystem studies of this type, it was a more complicated story than we had originally anticipated. I gave several presentations to interested people in the area.  A few times I had representatives from the chicken industry in the audience who challenged our data and interpretations. They had valid concerns. We had to trace the nitrogen from chicken feed to the Chesapeake Bay, which included eight different steps, all of which introduced uncertainties.
Seining for fish on the Nanticoke River
            We expanded our sampling to three major river systems on the Eastern Shore including the Nanticoke River, which contains the greatest concentration of chicken barns in its watershed. We sampled water, plankton, and small fish. The data showed—clearly to us in the know—that chicken waste nitrogen from uric acid ultimately made it into fish tissue. Whether that fact could be translated into a harmful algal bloom and fish kills was another problem. We never made that final connection because that year it rained a lot, and the concentrated chicken wastes were washed safely out to sea. We also had a problem with our sampling of leaves from shrubs and trees adjacent to the chicken farms.
Quinn and Marilyn found uric acid in the field
            The nitrogen isotope patterns in these leaves were the opposite from what we’d predicted. There was much less of the 15N isotope in the leaves within a radius about 1 kilometer around a chicken farm.  The following summer I worked with interns Allie Gale and Val Brenneis to capture ammonia, a gas that was wafting off of the piles of chicken manure. Everyone can identify with the smell of an animal feedlot. That smell is largely coming from ammonia (NH3).
Marilyn, Val Brenneis, and Allie Gale, teaching how to fish seine

There was a large isotope effect from the emanation of the ammonia from the solid chicken manure. We used the same techniques Wooller and I had perfected in our mangrove studies to collect ammonia in rainwater, storm drains, and the air. Contrary to what we’d originally hypothesized, the nitrogen from the chicken wastes had an additional pathway for spreading the pollution. The air surrounding the farms was laden with ammonia polluting a far larger region than direct application of chicken wastes to soil or into ground water.
Nutrients from chicken sh($ diagram
            The work’s not been published. Yet again, another story of biogeochemical complexity that we eventually figured out but have not formally put into the permanent record. I need to pull together the thousands of data points taken lovingly by my young colleagues and write a full on scientific report. Fortunately for the Chesapeake Bay, the Delaware and Maryland legislatures passed laws that helped the farmers deal with the wastes of the chickens they were growing to support the Big Ag chicken producers. Manure piles are now required to be covered and allowed to “ferment” on site. Farmers on the Eastern Shore never polluted the air and water intentionally, but had nowhere to dispose of the chicken wastes naturally generated. Near the end of our study, Perdue started a processing plant to receive the manure and convert it into a clean source of fertilizer that they shipped out to the Midwest where it was needed. Mat, Quinn, and I were given a special tour of the plant because we were recognized as scientists who were intent on understanding the problem, not necessarily out to “get them”. We laughed when we decided what to wear to tour a chicken shit processing plant: me, a nice blouse and slacks; Mat, a button downed shirt and tie; Quinn, a skirt and blouse.
Dressed to tour a chicken sh$# processing factory
            Pfiesteria blooms are, fortunately, no longer a major problem for the Bay.

Tuesday, November 12, 2019

Student Bobby J. Nakamoto—Mr. POM soon to be Dr. Fish

Bobby Nakamoto, UC Merced Castle Lab 2013

            “Are you his mother?” nurses asked in the emergency room of Mercy Hospital in Merced California. I was sitting in a chair across from my student Bobby Nakamoto, who was undergoing evaluation for what turned out to be a collapsed lung. His long dark hair was swept up in a surgical cap. It was a tense situation.
            “Uh—yeah, I guess so.” I answered, not knowing how to explain to these folks the complex relationship professors, in particular female professors, have with their long-term students. Bobby is half Japanese, half Caucasian. We look nothing alike. The medical people were a bit puzzled why I wasn’t sure how to answer.
            Earlier that morning, when Chris and I arrived on campus, Bobby was leaving the Science and Engineering building looking green. He and I had spent the end of the previous day diagnosing problems on the mass spectrometer. He appeared healthy at that time. On his way home twenty minutes later, he experienced chest pains and headed to the ER thinking he might be having heart trouble. Mercy Hospital is not at the forefront of medical care. After spending 8 hours with a chest x-ray and blood tests, Bobby was sent home with a “clean bill of health”. In the morning, the attending physician who had just come on duty noticed the x-ray showed Bobby had a collapsed lung and ordered him to come straight in. When I heard the news, without question I drove him to the hospital and spent the day as he suffered through several invasive procedures to treat his problem. Everyone needs an advocate when they are in the hospital. I was happy to take on that role. Finally, when I heard his father was on his way down from Northern California, I took off.
Bobby, UCM, New lab 2015
            I described earlier how I met Bobby in my first UC Merced Ecology class (see ** below). Since 2013, we’ve been working together and I’ve had the pleasure of watching him mature from a funny dapper undergraduate to a talented senior grad student who can fix a mass spectrometer, run complex statistical analyses on data, and digest the most complex ideas and theories. Along the way, I’ve asked him to take samples out of the dumpster, pick through coyote shit, work in the 105°F sunny, dry environments near UC Merced’s campus, and re-calculate hundreds of data points and write them down with pencil and paper. On the plus side, he’s also gotten in on the ground floor with hydrogen isotope amino acid measurements, something I originated previously and which will eventually have a substantial impact on the ecological field. He’s had a fully funded PhD project working with a collaborative team from UC Davis as well as UC Merced, and I’ve sent him around the world to present his work.
Marilyn Madness (l-r) Dan Toews, Marilyn, Jon Nye, Liz Wiliams, Joy Stewart, Bobby, 2016
            In return for the positive things, Bobby has provided my “eyes and ears” on the ground in the lab, when I am not there. His judgment in assessing a situation is pretty good for a young man of his age—almost 26 years old. As his major professor, I review his manuscripts and tests and encourage him when he has seemed overwhelmed. Recently, he had to write and submit a 15-page, double-spaced examination to qualify as a PhD candidate. He was required to do this without my normal editing and advice. For months, he moped around the lab. All of us in my lab group could see he was suffering from a lack of confidence, as though an albatross was hanging around his neck (see The Rhyme of the Ancient Mariner by Samuel Coleridge for the analogy). At the eleventh hour, I glanced at the penultimate version, added a couple of verbs to incomplete sentences, flagged a few commas, and said it looked great. With very few critical comments, he passed the exam on his own merit and hard work. What puzzles me sometimes is how such a talented person can have swings of feeling “not good enough.” I’m hoping that I’ll see continued progress in his learning, skills, and confidence as he passes through the next phase of his PhD work.
            Bobby’s research originally focused on using stable isotope tracers at the natural abundance level to learn about how organic matter—from both algal and bacterial sources—affects the food web in one of California’s river systems. The majority of rivers in California have been dammed to control flow for agriculture and to prevent disastrous flooding. Our research team, in which Bobby is a member, has been studying one of California’s undammed rivers, the Cosumnes that flows towards the San Joaquin-Sacramento Delta near the city of Stockton in Central California. Although the river has not been dammed, its banks have been levied with berms of soil to hold back floods during high rainfall events. Consequently, the ecosystem of levied and dammed rivers cannot and does not support the food web that it did historically, resulting in the collapse of some California’s native salmon populations.
            To understand how the Cosumnes river system is currently working prior to major earth-moving planned to remove some of the levies, Bobby collected and measured hundreds of samples of riverine “muck” filtered from one liter of water taken over the course of two years. We call the “muck” particulate organic matter or POM. Bobby has become Mr. POM of the Cosumnes, a title he’d rather not hold onto. His primary passion is for the study of fishes. While fishermen in bass boats ply the Delta bagging their limits of bass, our scientific team was unable to obtain a permit for any fish collection, because we might have accidently taken one of the endangered Delta smelts that are in severe decline, heading towards extinction.
Kingsley Odigie and Bobby, UC Riverside 2017
            Mr. POM took his hundreds of data points, graphed them, modeled them, and wrote his first paper where he took the lead on writing the document, compiling reviews, and submitting the paper. After more than 40 rounds of revision, the work is back in the hands of anonymous reviewers. Meanwhile, Bobby has his hands (well his analytical hands) on a set of Chinook salmon grown in experimental cages in rice paddies and the river. Working closely with a UC Davis team, he is figuring out whether “Dark Carbon” (not to be confused with Dark Matter) originating from microbial processes removed from photosynthesis is important for supporting the growth and reproduction of these native fish. Further, he’s branched out with a new collaboration studying fish and working with an evolutionary biologist from Texas A&M who has an interesting study system in rural Mexico. I’m thinking he’ll be moving on to the moniker Dr. Fish pretty soon.
            During the nearly seven years I’ve known Bobby, he’s had two serious relationships that I am aware of. As is typical with the difference in age and position, I don’t usually know what’s going on until after everyone else in the lab does. As a major professor and a science “mother,” there is a fine line between knowing too much about a person versus knowing just enough to keep someone on track to accomplish their work and be happy. For each and every student or postdoc, the path that I take is different for each, because, of course, we’re all individuals and no one size fits all. On that awful day in the hospital three and a half years ago, after two tries, the physician at Mercy Hospital managed to inflate Bobby’s lung. We were relieved. I could hang up my temporary science mother role for a while, and put on my more serious, demanding role as a Major Professor. I’ve got to see this young man launched and will work hard to see that I do.
L-r: Bobby, Jeanette, lab folks Riverside CA 2019
** “A second student who stood out that first semester was only a sophomore. On exam days, he wore a tie or a bowtie and dress pants. His first examinations placed him among the “A” students. His third midterm performance was sub-par. I wrote to the student, Bobby Nakamoto, asking him to come in for office hours. After a few days of not hearing from him, I called him to the front of the classroom after lecture and asked directly when he was coming into my office. It was the start of a six-year professor-student relationship that continues to this day.

            As my first semester wore on, I often looked to ecology students Bobby to explain simple mathematical concepts that some of the students weren’t grasping. He has a knack for explaining abstract concepts in simple terms, something I struggle with. Bobby worked as a volunteer for the following semester, then I hired him as a student assistant. He was assigned nutrient analyses of soils, meteorite extracts, and waters. Both of us think through procedures sequentially and abstractly. I could give instructions with a few words and a wave of my hands. He understood and carried out the work silently. When he was about to graduate, Bobby looked around at other graduate schools, but no one was smart enough to bring him into their program. He joined my lab group, now a senior graduate student, the person who can fix anything and is my right hand now that I’m physically limited.”

Rounding Third Base and Heading Home

Cards from Franny and Flowers the Rumbles   My daughter Dana is marrying George Goryan on June 25 at our home in Mariposa...