MycoTour and Reinoculation

Striking the correct balance between keeping this blog up-to-date and posting information that is interesting to even the most avid mycoremediation devotee has been challenging. Although a lot of my time was recently spent preparing for and recovering from our 11-day MycoTour of Ecuador. From the previous entry one might infer that it was a complete disaster. However, I want to say that while Bill’s death was hard on everyone, we had an unforgettable time together and learned a lot. The MycoTour brought together mushroom aficionados from mycological societies from as far south as San Diego to as far North as Vancouver BC. The West Coast fungivore community was well represented.

With our fantastic group we visited our field sites and heard about the current developments in the Texaco-Chevron litigation from Frente de Denfesa de la Amazonas lawyer Pablo Fajardo. While at the Frente offices, we had the opportunity to view the short documentary entitled Contamination and Testimonies of the Affected which you can view on Frente’s website. Later in the tour we had the opportunity to apply the collective knowledge of the assembled mycophiles to aid an ongoing biological inventory at the research refuge BioMindo in Mindo, Ecuador. AMP volunteer Danny Newman has posted a collection of the his excellent photos of the scores of mushrooms we encountered on our forays and at our field sites here.

All this activity might cause one to wonder what has been happening with the research. Backing up a bit, just before the MycoTour Ricardo and I went out to Lago Agrio to add mycelium to our boxes and pits. Some of our treatments had fresh mycelium replacing old, dying mycelium and some of our treatments had the old mixed with the new. As it turns out, we got overconfident with our excellent growth and did not anticipate that the turning would risk contamination. So our pits have been infested with an opportunistic black mold. This isn’t disasterous, but it is an opportunity to learn about the best way to maintain the strongest mycelium possible.

We decided to reinoculate with fresh mycelium to continue our culture, since all fungi lives only so long on a given substrate. If reinoculation is not a smart option, then playing with our initial inoculation and it's proportion to the contaminated soil is a good option. Our use of sawdust means that our mycelium has a shorter, but more vigorous life than if it were growing on say, cut logs and branches. The reason for the shorter life-cycle on sawdust has to do with the greater surface area offered by sawdust. The more surface area, the greater the biological availability of nutrients and, in our case, the quicker the remediation.

The current set of experiments has had a greater focus on qualitative assessment, punctuated by modest chemical analysis to compare with our observations. Our reasoning for this had a lot to do with budgetary concerns, since chemical analysis is an expensive process and often not necessary to establish that a fungal culture is healthy. Basically, with the information we have gleaned in the last six months of experimentation, I feel confident that we can design other, smaller and shorter experiments that will help us fine tune our method.

In other news, in less than a month Jess and I will be visiting the United States along the West Coast and the Chicago area. Watch this space for speaking dates and feel free to email me if you would like to host us at your local mycological society, classroom or community center.

Farewell to Bill

Bill and Joan joined us for our very first MycoTour of Ecuador as part of a fundraiser for our work here. The MycoTour included visits to our field sites in Lago Agrio, mushroom forays in Ecuador's jungle and, among many other activities, a reality ''Toxic Tour'' of oil contamination in the Sucumbios province. Both mushroom enthusiasts living in Vancouver, B.C., Bill and Joan were a delight to travel with. We were all shocked by Bill's sudden passing, as we were all impressed by the easy pull-ups that this tough, friendly man was doing in the jungle lodge we visited. I feel lucky to have shared Bill's 83rd birthday in the Amazon with him and honored to have known such a vigorous spirit as Bill. Both Jess and I count Joan as one of our good friends and our heart goes out to her in this difficult time. Below I have reposted Bill's obituary, may he rest in peace.

William Campbell

It is with great sadness that we announce the sudden and unexpected death
of William “Bill” Campbell, in Papallacta, Ecuador on July 18th, 2008, two
days after his 83rd birthday. Bill was born in Derry, North Ireland and raised
in Glasgow, Scotland, served two years in the British Army and married
Margaret McGoldrick Doyle in 1952. They lived in Paisley, Scotland where he
still has many friends and relatives today. Bill and Margaret came to live in
Canada in 1966 and chose White Rock, BC to be their home since 1969.

Bill was pre-deceased by his wife of 47 years in 1999 and a son, John, in
1975. He is survived by his son William “Liam” Campbell of White Rock,
BC, step-sons Joe Doyle of Richmond B.C. and Jim (Kath) Doyle of Oldham
England and their family. As well, a sister, Lilly Conway and family and
brother, John (Isabelle) and family in Scotland. Bill will be deeply missed by
his close friend and companion of 8 years, Joan O’Reilly and her children
and grand children. At the time of his untimely death, Bill and Joan were
travelling with the Amazon Mycorenewal Project in Ecuador. Special thank
you to the coodinators of the project, especially Jess Work and Brian Pace
who provided an incredible amount of assistance, support and translation
when dealing with the authorties to arrange creamation to bring Bill home.
Prior to leaving Ecuador, some of Bill’s ashes were spread on the Island of
Isabela in the Galapagos, a place Bill always wanted to go.

A funeral service will be held at the Star of the Sea Parish, Good Shepherd
Church, 1:00 p.m. Wednesday, August 6th, 2008 located at 2250 150
Street, Surrey. A celebration of Bill’s life will follow at the Elks Lodge #431,
1469 George Street, White Rock. Internment will take place at Gardens of
Gethsemani.

If people wish, in lieu of flowers they
may send donations in Bill’s name to
the Amazon Mycorenewal Project, a
volunteer based organization aimed at
cleaning up oil polluted areas in Lago
Agrio, Ecuador. Please contact Jess
Work via email jesswork@gmail.com
for further information and to donate to
this project.

Caterwaul Quarterly AMP Article

There is plenty going on down here in Ecuador, but I have been taking a much needed short vacation. I will be writing a more full account of our reinoculation and latest field observations in the next few days, but for now here is a link to an article I wrote about our project in a new online periodical called Caterwaul Quarterly.

Vivan los Hongos!

On May 28th, community representatives from Ecuador and Nigeria are traveling to San Ramon, California along with Bay Area activists to demonstrate at the gates of Chevron corporate headquarters during their annual shareholder meeting. Supportive shareholders inside will confront Chevron executives about the toxic legacy of oil extraction in the Ecuadorian Amazon. The Nigerian delta has also faced similarly serious health and ecological consequences as a result of oil development.


As for news on the fungal front, I am quite happy to write that our second attempt at mycoremediation in the Amazon has quite literally born fruit. Three weeks after we set up our experiment, a number of the boxes have begun to fruit mushrooms—directly out of the petroleum-saturated layer. We put yellow plastic screens directly on top of the contamination layer for easy monitoring. When we need to take a sample, we can simply lift the screen and get down to the area of interest. The screens also have the benefit of providing a clear visual marker for locating the oil layer.




Multiple clusters of little mushroom primordia (baby mushrooms!) were poking out everywhere from fuzzy-white mycelium. In many boxes, the overall effect was a graying of the black, oil-soaked sections as a result of dense mycelial growth. Every single box currently has healthy mycelium growing inside the contaminated layers, even in our most concentrated treatments, such as the one pictured.



There are no mushrooms fruiting in the large pits we dug and filled with spawn, sawdust and other mushroom substrates. However, the lack of fruit bodies is really not important, since the vegetative state of the mushrooms, the mycelium, is what does the majority of the heavy-lifting in remediation. By use of the yellow screens we were able to peer under several feet of piled spawn and substrate and confirm the presence of mycelium in our pits as well. These fine threads of fungi branch and grow densely through soil and substrate, excreting oil-digesting enzymes along the way.

Visualizing the growth processes in action here is an exercise in pretending to be very small. If I was roughly the size of a cell, the span of a centimeter or two is quite daunting. This is the scale that mycelium operates on. Our mycelium responded to a similar distance created by the plastic screens by sending down ropey rhizoids, root-like fungal structures used for entering a substrate. We can assume that the fungus produced rhizoids because it recognizes petroleum as a tasty meal and is willing to expend metabolic energy to reach (pictured).

In some of our pits, we encountered some weedy fungi that rapidly fruited and died. Despite this, directly under these mushrooms was healthy, almond-scented oyster mushroom mycelium. As such, it seems that our oyster mushrooms are able to hold their own against the first round of competition.

In the following weeks we will be taking samples for laboratory analysis to get a quantitative picture of exactly how much hydrocarbon pollution has actually been digested. Qualitatively speaking, our progress thus far has met and exceeded all of our highest expectations.

photo dump

Oil-acclimated sawdust spawn

Redefining volunteerism in the Amazon

Three different contamination concentrations

Here we have some low-tech pastuerization of mushroom substrate

The hair mats, in all their glory

Four deep, plastic-lined pits, ready for action

In situ remediation site

Cooking with Crude

The big news around Lago Agrio is that Ecuadorian lawyers Pablo Fajardo Mendoza and Luis Yanza each won the coveted Goldman Environmental Prize for their work building a class-action environmental lawsuit against Texaco/Chevron for what is now an estimated $16 billion in damages. It was an important time for us as well, since we were busy setting up our second mycoremediation experiment on Donald's land. Drawn by the news of the Goldman Award, two journalists dropped by to snap photos of our work throughout our stay. It also seems that folks in Fort Bragg, CA are also looking to take the mycoremediation out of the lab and into the field to work on dioxin pollution.

I have to take this opportunity to say that if it were not for the outstanding effort of our six volunteers, two competent hired hands, Donald, Jess and Ricardo, I'd still be in Lago. Thanks, guys. Working with crude oil is just about the messiest job I can think of, and we all spent 7 long, hard days getting to know a barrel of the stuff.

We collected the crude from a 30 year-old waste pit because we wanted to test our method with the nastiest petroleum contamination we could find. The waste pit we collected from was smack in the middle of jungle land. Despite the dense thicket of Ecuadorian Amazon that closed around all sides, the contamination had effectively created a clearing in the canopy where trees and underbrush were unable to grow. Only a few sickly, scrubby plants clung to life on top of the litter that had built up over the decades on top of the aging crude. We built a bamboo bridge of sorts to stand above the pool and formed a chain of goop-filled buckets back to the truck that held an empty 55 gallon barrel.

On the way back to our site, we stopped at a sugarcane farm and drank some of the sweet, green juice being produced for raw sugar. Below the horse-powered sugarcane mill was a large field of sugarcane husk. We collected a small load of the sugarcane husk, thanked our hosts and moved on. The husks represented an abundant potential mushroom substrate and we intended to include it in this round of investigations. During this trip, we also collected coconut shells to use the discarded fiber and local grass-straw for more substrate trials. And of course, who could forget the human hair. As it turns out, some of our friends thought our cause was worthy enough to donate their dreadlocks and one of our volunteers, Eric, was even inspired to lend his beard to the project.

The barrel of crude had to be processed a bit before we could begin our experiment. Old oil off-gases toxic volatile compounds for years and some of it eventually hardens into an asphalt-like substance. Those asphalt stones represent a problem for our analytical purposes because they are 100% petroleum and cause the lab to report much higher levels of petroleum than are biologically significant. In a real-world remediation project, we would not have to worry about them since they have essentially become part of the local geology at that point and are biological-nonparticipants, inaccessible by hyphae and otherwise not really going anywhere. This is a long way of saying we had to push a barrel of gloppy oil through a metal sieve by hand. It only took a day and a half of three people in safety gear working nonstop.

Next came the preparation of our contamination mixture. By carefully measuring how much petroleum we had at the beginning of our test, we are able to determine the total amount and rate of hydrocarbons being metabolized by our mycelium throughout our experiment. While also mostly unnecessary in an applied mycoremediation scenario, this meant mushing buckets of earth with oil and sawdust in various proportions by hand. It was kind of like making really disgusting bread. Knowing these ratios also provides us with information important for assessing how much earth and/or organic material must be added to a given site to create a comfy environment for our mushrooms to do their work. Always thinking low-tech, we heat-pasteurized our substrate in 55 gallon drums over both gas-fueled and wood burning fires. The acclimated mycelium had already colonized bags full of sawdust and by adding them to pasteurized bulk substrate, we allowed them to expand their mycelial mass quickly in a new environment rife with potential competitors.

Since the last thing any of us wanted to do was create more pollution by spreading the existing stuff around, we took every precaution in the protection of Donald's land. While a large number of our experimental treatments are being conducted in small microcosm boxes, we tested larger areas more similar to actual waste sites in very large, deep pits. Somehow, the exciting world of mycoremediation required me to dig holes in the dead Amazonian heat for three days. The pits were lined with two layers of the thickest plastic available and staked into place. Inside, we heaped contaminated soil, substrate and acclimated mycelium for remediation. In two weeks we will be checking our progress, but Donald is there monitoring the site daily.

Mycorenewal and Ecology

Our mycelium is growing vigorously in the lab. It has been acclimated to metabolizing oil and seems healthy and strong. This is good news of course, but ours is a question of ecological engineering as well as laboratory wizardry. Even though our pet organism is doing well in the Petri dish, this has no bearing whatsoever on how well it does in the field. There is a big difference between life in the Petri dish and the Amazon. So will our oysters become food for insects or out competed by other weedy fungi? We don’t know. However, Pleurotus (oyster) mushrooms happen to be among nature’s most aggressive fungal species, metabolizing everything from wood to coffee grounds to, well, oil. Their enzymes have shown to experimentally breakdown Polychlorinated biphenyls (PCBs) and somehow all fungi have to find a way to compete in a hazardous environment when the vast majority of their biomass consists of threadlike appendages only one-cell thick. So how do they do it?

The long answer is an adventure into complexity or in other words, ecology. The short answer is we don’t know. Well, not exactly. Soil is alive. It is chalk full of critters large and to be sure, small. Really small. Bacteria, lots of bacteria, fungi, spiders, mites, beetles, nematodes, plants small mammals and reptiles all vie for carbon and nutrients in every cubic meter of soil. So why would fungi ever be able to build their vast networks under our feet if they are constantly hectored by hungry, exponentially growing bacteria along every centimeter of their hyphae? The answer of course is, we don’t know, not really.

However, we do have some ideas. Symbiosis is quite frequent throughout nature. In fact, it is happening in every cell of every creature on the face of the planet that has a nucleus. It happens to be most common across Kingdoms, that is organisms that are vastly different from one and other in form and, importantly, metabolism. So some scientists theorize the that fungi form symbiotic relationships with specific, friendly bacteria that keep other, unfriendly bacteria at bay. Unfortunately, the Law of the Jungle in the jungle is somewhat erroneous. Tooth and claw have their role to play in every ecosystem, but cooperation and mutualisms are far to common to ignore.

Plant-fungal symbiosis is as ubiquitous as it is ancient. For over 400 million years—since plants first colonized land—some 90% of all plants have formed fungal partnerships inside or around their roots. Plants gain access to difficult to reach water and nutrients sources and protection from disease while the fungi, called mycorrhizae (“root-fungus”) receives sugar manufactured by the plant. It is possible that different species of fungi form mutualistic relationships with bacteria where select species of beneficial bacteria are encouraged to grow in the area surrounding the mycelium, providing protection in exchange for some nutrient currency. An arrangement such as this is not hard to imagine, in fact, it seems we humans have a similar arrangement in via our appendix: recent findings have identified the appendix as a bacterial safe-haven for beneficial bacteria that help keep pathogenic bugs at bay.

While the ecology of symbiosis can help clarify the niche of fungi in the environment, we need a different take on ecology to see why we are confident that our oysters will have a decent chance of thriving in their new home. All living things are capable of bearing offspring at a rate that far outpaces mere replacement. Usually what keeps any one organism from taking over the world with offspring in a few generations are predators, diseases, famine, suitable habitat and natural catastrophe of all scale. We are designing our project to give our acclimated oyster mushrooms a leg up by taking advantage of what is known as an ecological desert created by the petroleum contamination. The areas are toxic by definition because no other organism in any significant numbers likes to munch on oil. Therefore, we would expect a life form specifically trained (not, to be clear, genetically engineered) to enjoy such a meal would have a distinct advantage in such an environment and reproduce prolifically.

Creating an ecological advantage for our mushrooms will require more than just sowing it over a lagoon of oil. While the oil-digesting capabilities of oyster mushrooms may be a part of the new technology of mycoremediation, the specific process of its application to an affected area is what will ultimately allow it to succeed with as little input and maintenance as possible. This is important because the area contaminated in the Ecuadorian Amazon is so vast and heterogeneous that our techniques will likely have to be site-specific. We will be back in Lago Agrio by mid-April to begin our new round of trials. It is our hope that our latest experiment should provide a baseline methodology that will serve as a template for other Amazonian sites blighted by ill-disposed petroleum waste.

The Amazon Mycorenewal Project is currently looking for additional NGO partners with complementary missions to help support our work here in Ecuador. Interested organizational representatives should contact me at brianapace@gmail.com for more information about our mission and work. Also, Amazon Mycorenewal Project has a new website:

www.amazonmycorenewal.org

and

www.kallambas.org



mycorrhizae photo from: www.helsinki.fi/bioscience/myco/