Thursday, September 3, 2015

Fuels and fungi: A love story

Fuels and fungi have a long-standing and intricate relationship. Before we get into the juicy details, let's examine the players.

Traditionally speaking, a fuel is any organic substance (e.g. wood, coal, and oil) containing lots of stored chemical energy within the bonds holding its atoms together. This energy can be released via combustion, which we harness to move cars forward and generate electricity.

Fungi are a huge group of organisms largely describable in terms of what they lack: locomotion and the ability to use the sun or electron-rich rocks to make their own food. They tend to form networks of many-celled filaments (exception: yeasts, which are generally unicellular) and reproduce by churning out tonnes of spores. In addition to eating human foods, as well as humans themselves, fungi have specialized in accessing the energy contained within organic bits (e.g. wood, feathers, nails, and plastics) recalcitrant to the efforts of most other organisms.

Recycling wood

Wood is a good starting point for talking about fuel-fungi interactions. It's the hard tissue produced by a group of plants affectionately known as trees, densely packed with energy-rich organic carbon (mostly lignin and cellulose) we get at by burning it in stoves or campfires. Most organisms lack the ability to digest wood and kick-start the return of its nutrients to the soil, but fungi have it figured out. A good thing, too, since otherwise dead trees would just pile up everywhere as they did prior to the evolution of wood-eating fungi about 300 million years ago.

Wood is consumed by a large group of fungi going by names like white rot and brown rot. These terms refer to what the fungi leave behind after consuming the brown lignin or white cellulose, respectively, out of wood. Lignin encompasses a large group of intricately linked aromatic alcohols involved in movement of water throughout trees. Woods with lots of lignin are usually highly durable and make good fuel since lignin contains more energy than cellulose. Used to make paper and cardboard, celluose is a bunch of glucose molecules chained together in a particular way (just a slight tweak in how the chain is linked and you get starch, accessible to way more organisms). Cellulose can also be converted into biofuels using fungi (more on that later).

A scrumptious Laetiporus sulphureus eating the cellulose out of wood (Source)

Many well known edible mushrooms eat wood, such as chicken of the woods, oysters, honeys, and shiitakes. Some munch on a tree while it's still alive, while others respectfully wait until it dies. To break down the lignin and cellulose in wood, fungi often produce hydrogen peroxide, a strong oxidizer used as a propellant in rockets. So there's another fuel-fungi link.

Enabling cow farts

Anaerobic fungi are found in the gastrointestinal workings of ruminants (e.g. cows), where they turn plant matter and other ingested materials into hydrogen gas and simple organic acids (e.g. acetate, propionate, and butyrate). These products, in turn, support the growth of neighbouring bacteria and archaea capable of making methane, the principal component of natural gas and known fuel in its own right. Unsurprisingly, gut fungi are particularly good at breaking apart the cellulose present in fibrous plants, facilitating their digestion and conversion to methane.

Cleaning up coal

Fungi can also live off of geologically processed dead organisms, perhaps better known as fossil fuels. Coal is formed from dead plant matter, which is progressively compacted into denser (and thus more energy rich) materials over time (peat -> lignite -> bituminous coal -> anthracite).

While we've been progressively using up the Earth's supply of easily obtainable higher rank (more energy dense) coal, lots of poor quality coal remains in the ground. This crap coal is highly polluting and has a relatively low energy content, so some researchers have proposed using microorganisms to convert it into more environmentally friendly methane. This is accomplished by employing fungi (e.g. Penicillium chrysogenum) to leach complex organic molecules from the coal matrix and break them down into simpler forms, which are then fed to anaerobic bacteria and archaea to produce methane.

A chunk of crappy brown coal (lignite) (Source)

Fungi can also be used to clean up poor quality coal by removing organic sulfur contained within it. This is important because it reduces the amount of sulfur dioxide (acid rain precursor) released when the coal is combusted. Biodesulphurisation of coal has been demonstrated using the white rot fungus Phanerochaete chrysosporium.

Munching on jet fuel

In addition to coal, fungi can eat crude oil and liquid hydrocarbons (e.g. gasoline) derived from fossil fuels. This is problematic since they can end up growing inside places like oil refineries, fuel storage tanks, and vehicle fuel tanks. They typically reside wherever water (even small amounts arising from condensation) comes into contact with fuel (e.g. a small centimeter-thick layer of water at the bottom of a fuel tank). Not only will fungal growth potentially reduce the quality of a fuel, but organic acids secreted by fuel-contaminating fungi can corrode metal parts in contact with the fuel (e.g. diesel storage tanks made out of carbon steel) and fungal biomass can clog up valves, gauges, fuel lines, and filters.

In the 1950s, the US Air Force ran into problems with fungi in fuel tanks. For a brief while they had to cut back on some of their flight operations because aircraft fuel control systems and refueling equipment weren't working properly. In 1958, the crash of B-52 bomber was traced back to a clogged in-line fuel filter. Subsequently, improved cleaning efforts and the introduction of new fuel additives effectively reduced microbial growth in fuel systems.

In the field (i.e. storage tank, refueling truck, aircraft wing tank), aviation fuel often contains mixed bacterial and fungal communities including many filamentous fungi (moulds) and yeasts commonly found in the natural environment (e.g. members of the genera Acremonium, Aspergillus, Candida, Fusarium, Penicillium, and Rhodotorula).

Specific fungi known to dine on liquid hydrocarbon fuels include Cladosporium resinae (the so-called kerosene fungus, responsible for green-brown slimes at the bottom of fuel tanks all over the world), Monascus floridanus (initially isolated from the resin-soaked roots of a pine tree endemic to the southeastern US) and Yarrowia lipolytica (found in cheese, sewage, and spilled crude oil - wherever lipids are available for consumption).

Making food from fuel

In the 1960s, oil companies happened upon the grand idea of using crude oil to produce lots of edible yeast. For example, this US patent outlines a process whereby Candida tropicalis can be cultivated using a water-based nutrient medium to which has been added a crude oil derivative consisting mostly of straight chain hydrocarbons. The yeast is then chemically extracted from the resulting gunk and somehow sold as food. Hooray!

Incidentally, yeast has a pretty decent history as a food. Torula (Candida utilis) rose to prominence during World War II as countries looked for a means to address wartime shortages in food and animal feed. Nutritional yeast (usually Saccharomyces cerevisiae) is a cheese substitute often found in natural food stores. Finally, marmite and its ilk are made from the guts of dead yeast cells left over from brewing beer.

To crop, or not to crop (Source)

Apparently the numbers, when crunched, suggested it was possible to produce about 20 million tons of yeast protein from crude oil every year. At the time, petroleum was a cheaper raw material than plant-derived carbohydrates and also produced relatively greater yields of yeast. Tragically, the initiative never made it off the ground. People (understandably) weren't down for eating yeast made from the same stuff used to make gasoline and it proved difficult to isolate the yeast from toxic hydrocarbons present in crude oil.

Making fuel from food

Looking in the other direction, yeasts of the genus Saccharomyces are famously used to convert plant matter and water into ethanol-rich liquids. While many of these are imbibed for pleasure, others are chemically processed to isolate the ethanol for use as a fuel. This is nice because then we don't have to use fossil fuels, of which there are a finite supply. When used to power a vehicle, ethanol is typically mixed with gasoline, although engines capable of running on pure ethanol are a thing as well. Classic yeast-driven ethanol production proceeds as follows: (1) Starch-rich plant bits (e.g. sugarcane juice, grains such as corn and wheat) are mashed up and cooked to release the starch, (2) Enzymes are added to break the starch down into glucose, (3) Yeast (usually a strain of Saccharomyces cerevisiae) is added to convert the glucose into ethanol, and (4) Ethanol is recovered via distillation and dehydration.

Some argue ethanol isn't an ideal fuel since it has a relatively low energy content and many current engines aren't designed to use it. Fortunately, fungi (along with algae and bacteria) can can also be used to make larger hydrocarbons (e.g. various lipids and alcohols collectively known as biodiesel) more amenable to fuel use (i.e. higher energy content, closer resemblance to currently used fuels). For example, filamentous fungi (moulds) such as Mucor circinelloides and Syzygites megalocarpus are capable of churning out highly nutritional oils.

Oils are also put together and highly accumulated by several yeast species. These yeasts can be grown to high densities within specially designed chambers (bioreactors), accumulating up to 70% oil by dry weight. Relative to algae, which are being widely researched as a means of producing biodiesel, yeasts can potentially grow faster and to higher densities, and are less susceptible to viral infections or bacterial contamination (since they can be grown at low pH).

In addition, potential fuel producers have been found among fungi living inside plants (endophytes). They produce volatile organic compounds such as 3-methyl-1-butanol with fuel potential, and as an added bonus, can do so when grown on plant-based agricultural wastes rich in cellulose (e.g. beet pulp, corn stover, sugarcane bagasse) (vs. the stricter high-starch requirements of ethanol making yeasts). The ability of these fungi to grow on plant wastes likely reflects their status as the first decomposers to access a plant after it dies (seeing as they're already hanging out inside it).

Poisoning foragers and powering rocket ships

Hydrazine and its derivatives, monomethylhydrazine (MMH) and 1,1-dimethyl hydrazine are highly toxic, likely carcinogenic, super flammable, and explosive. They also have been used to power components of the Space Shuttle, a couple of terrifying intercontinental ballistic missiles, and the F-16 fighter jet.

The fungus Gyromitra esculenta forms a morel-like mushroom and is super poisonous unless prepared correctly. This toxicity is due to gyromitrin, a volatile and unstable metabolite that is readily transformed into MMH. Once the mushroom is eaten, the acidic conditions of the stomach ensure a rapid conversion to MMH. Cudonia circinans, like G. esculenta, contains a lot of potential MMH. Both fungi are found growing beneath conifers, but while G. esculenta has a brain-like reddish brown cap and appears in the springtime, C. circinans has a yellow cap and shows up in late summer and fall. So ends our brief field guide.

There's some fungus metabolite in there somewhere (Source)


Andary C, Privat G, Bourrier MJ. 1985. Variations of monomethylhydrazine content in Gyromitra esculenta. Mycologia 77(2):259-264. [First page]

Arshadi M, Nilsson C, Magnusson B. 2006. Gas chromatography-mass spectrometry determination of the pentafluorobenzoyl derivative of methylhydrazine in false morel (Gyromitra esculenta) as a monitor for the content of the toxin gyromitrin. Journal of Chromatography A 1125(2):229-233.

Buddie AG, Bridge PD, Kelley J, Ryan MJ. 2011. Candida keroseneae sp. nov., a novel contaminant of aviation kerosene. Letters in Applied Microbiology 52(1):70-75. [Full text]

Farmwald JA, MacNaughton MG. 1981. Effects of hydrazine on the activated sludge process. Journal (Water Pollution Control Federation) 53(5):565-575. [First page]

Favaro L, Jooste T, Basaglia M, Rose SH, Saayman M, Görgens JF, Casella S, van Zyl WH. 2013. Designing industrial yeasts for the consolidated bioprocessing of starchy biomass to ethanol. Bioengineered 4(2):97-102. [Full text]

Gonsalvesh L, Stefanova M, Marinov SP, Carleer R, Yperman J. 2014. Geochemical study of maltenes from coal biodesulphurisation. Fuel 135:332-339.

Haider R, Ghauri MA, SanFilipo JR, Jones EJ, Orem WH, Tatu C, Akhtar K, Akhtar N. 2013. Fungal degradation of coal as a pretreatment for methane production. Fuel 104:717-725.

Hendey NI. 1964. Some observations on Cladosporium resinae as a fuel contaminant and its possible role in the corrosion of aluminium alloy fuel tanks. Transactions of the British Mycological Society 47(4):467-475.

Humphrey AE. 1967. A critical review of hydrocarbon fermentations and their industrial utilization. Biotechnology and Bioengineering 9(1):3-24.

Kittelmann S, Naylor GE, Koolaard JP, Janssen PH. 2012. A proposed taxonomy of anaerobic fungi (class Neocallimastigomycetes) suitable for large-scale sequence-based community structure analysis. PLoS One 7(5):e36866. [Full text]

Mountfort DO, Asher RA, Bauchop T. 1982. Fermentation of cellulose to methane and carbon dioxide by a rumen anaerobic fungus in a triculture with Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri. Applied and Environmental Microbiology 44(1):128-134. [Full text]

Nicaud JM. 2012. Yarrowia lipolytica. Yeast 29(10):409-418. [Full text]

Rauch ME, Graef HW, Rozenzhak SM, Jones SE, Bleckmann CA, Kruger RL, Naik RR, Stone MO. 2006. Characterization of microbial contamination in United States Air Force aviation fuel tanks. Journal of Industrial Microbiology and Biotechnology 33(1):29-36. [First two pages]

Sekhohola LM, Igbinigie EE, Cowan AK. 2013. Biological degradation and solubilisation of coal. Biodegradation 24(3):305-318.

Sitepu IR, Garay LA, Sestric R, Levin D, Block DE, German JB, Boundy-Mills KL. 2014. Oleaginous yeasts for biodiesel: Current and future trends in biology and production. Biotechnology Advances 32(7):1336-1360.

Strobel GA. 2015. Bioprospecting—fuels from fungi. Biotechnology Letters 37(5):973-982.

Vasilyeva AA, Chekunova LN, Bilanenko EN, Kachalkin AV, Polyakova AV. 2012. Characterization of the strain Monascus floridanus PF Cannon & EL Barnard, isolated from aviation fuel. Microbiology 81(2):244-250.

Wiley AJ, Dubey G, Lueck B, Hughes L. 1950. Torula yeast grown on spent sulfite liquor. Industrial & Engineering Chemistry 42(9):1830-1833. [First page]

1 comment: