Thermophilic Fungi for Industrial Bio-Methane Production

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This entry is adapted from the peer-reviewed paper 10.3390/microorganisms11102600

The novel industrial approach of co-fermenting wood wastes with agricultural wastes that are rich in nitrogen such as animal manures to produce bio-methane (renewable natural gas) fuel via thermophilic anaerobic digestion mimics an analogous process occurring in lower termites, but it relies instead on thermophilic fungi along with other thermophilic microorganisms comprising suitable bacteria and archaea. Wood microbial hydrolysis under thermophilic temperatures (range of 55 °C to 70 °C) and aerobic or micro-aerobic conditions constitutes the first step of the two-step (hydrolysis and fermentation) dry thermophilic anaerobic digestion industrial process, designated as “W2M3+2”, that relies on thermophilic fungi species, most of which grow naturally in wood piles. Eleven thermophilic fungi have been identified as likely agents of the industrial process, and their known growth habitats and conditions have been reviewed.

fungus hydrolysis fermentation thermophilic wood bio-methane

1. Introduction

Woody biomass represents an abundant renewable resource that is carbon-neutral by design, and consequently, it has been a topic of intense interest as a source of renewable energy. However, the traditional approach of the combustion of wood to extract energy in a large scale is no longer considered to be an environmentally viable choice ^[1]. Thus, other pathways for the utilization of woody biomass must be developed. One such pathway is based on the industrial replication of naturally occurring processes observed in lower termites ^[2]. This pathway has been converted into an industrial process designated as “Wood to Methane 3+2” and abbreviated as “W2M3+2” ^[3]^[4]. This process consists of the microbial fermentation or anaerobic digestion of wood wastes into bio-methane as an advanced transportation fuel with a carbon-negative footprint, along with the generation of other co-products such as commercial grade phosphate, nitrogen and potassium bio-fertilizers, and green carbon dioxide to be used as either an industrial gas or a feedstock for other chemicals and fuels including additional or secondary bio-methane production with the aid of hydrogen generated via water electrolysis using wind and solar power. A major departure of the industrial conversion process from the natural one occurring in lower termites is that the former is designed to operate in the thermophilic temperature range of 55 °C to 70 °C, unlike the latter, which is of a mesophilic nature with optimal operating temperatures below 40 °C. Thus, a different set of microbes, namely, thermophilic fungi, is employed to promote the fermentation of wood. Otherwise, the industrial process is quite analogous to the natural one that is observed in lower termites.

2. The Thermophilic Fungi of Choice for Bio-Methane Production

Wood size reduction is a key step in the decomposition of wood in the natural process and in the industrial process ^[3]^[4]. Wood particles of 0.2–2 mm in size are typically generated in the industrial process, whereby the cellulose crystallinity is disrupted. Moreover, the hugely increased surface area of wood immensely facilitates the exposure of cellulose and hemicellulose to the enzymes secreted by the thermophilic organisms that promote wood hydrolysis ^[5]^[6]^[7]. The hydrolysis process is driven primarily by thermophilic fungi ^[8]^[9]^[10], while thermophilic actinomycetes synergistically assist the fungi in the process ^[11]^[12]. In addition, about 20% to 30% of the nitrogen in the manures exists as ammonia, thereby enhancing the break-down of the wood particles by facilitating the solubilization of lignin in the “W2M3+2” industrial process ^[5]^[13]^[14].

Thermophilic fungi constitute the major component of the microflora that develop in heaped masses of plant material, piles of agricultural and forestry products, and other accumulations of organic matter, wherein a warm, humid, and mostly aerobic environment provides the basic conditions for their development ^[9]^[15]. The typical temperature and air distributions obtained in such an environment, reflected in a mushroom compost pile, is shown in ^[16].

Thermophilic fungi comprise only a very small number of species on the order of 30 to 40 out of about 50,000 recorded fungi species ^[13]. They have an optimum temperature for growth above 45 °C. Moreover, thermophilic fungi are the only known eucaryotic organisms to function in temperatures above 50 °C and perhaps as high as 70 °C ^[9]^[16]. Of course, the property of thermophily is much more widespread among other unicellular organisms such as blue-green algae (85 °C), actinomycetes (85 °C), and archaea (110 °C). Thermophily in fungi is obviously not as extreme as it is in eubacteria and archaea. People should also note that in general, the term of thermophily does not refer to a specific temperature that is applicable to all organisms, but rather to a temperature range that is applicable to a particular species, although it has a lower bound of 40 °C. In the first two decades of the 20th century, the number of studied thermophilic fungi species had risen to five, and toward the middle of the 1960s, it rose to thirteen species ^[9]. As it turns out, thermophilic fungi have been essentially observed since the discovery of their existence because of an interest in their activities regarding practical applications rather than because of trying to understand their taxonomy and morphology ^[16]. Thus, in the past three decades, researchers have isolated a dozen or so thermally stable enzymes out of about two dozen species of the known thermophilic fungi to utilize these enzymes in a variety of biotechnological applications ^[16]. The employment of thermophilic fungi in the hydrolysis of wood wastes to produce bio-methane is therefore no exception to the trend.

Because of the way thermophilic fungi have been discovered and studied for more than a century now, and particularly in the past half-century, there seems to be a confusion as to the true species of thermophilic fungi versus thermotolerant fungi ^[16]. Moreover, there exist variations among several species of thermophilic fungi with respect to their reproductive nature (sexual vs. asexual) that add further confusion to their classification and make the nomenclature even more cumbersome ^[16]. This also results in nomenclatural disagreements ^[15]. Nonetheless, people have been able to identify at least eleven thermophilic fungi species that can operate optimally at a temperature of 55 °C and possibly up to 70 °C. These fungi are found naturally in habitats where wood wastes, other cellulosic wastes, and manures exist ^[16]^[17]. These fungi then are of exceptional importance in the industrial wood hydrolysis and are the drivers of this process, and they are aided synergistically by thermophilic actinomycetes that are tolerant to even higher temperatures. A summary of these eleven thermophilic fungi species, along with their optimal environmental temperatures, pH conditions, and respective natural habitats.

In the wood hydrolysis step of the industrial operation, “W2M3+2”, several of these species of thermophilic fungi are believed to operate synergistically along with thermophilic actinomycetes to break down cellulose and hemicellulose into simple sugars, depending on the specific compositions of the processed wastes. For example, Thermomyces lanuginosus, also named or known in earlier studies as Humicola lanuginosus, which, by itself, cannot metabolize cellulose, has shown profuse growth in mixed cultures with the cellulolytic fungus, Chaetomium thermophile ^[10]^[18]. Another vigorous cellulose-degrading fungus is Thermoascus aurantiacus. In fact, all eight thermophilic fungi species that grow on wood chips (Chaetomium thermophile var. coprophile, Chaetomium thermophile var. dissitum, Humicola grisea var. thermoidea, Humicola insolens, Scytalidium thermophilum, Talarmyces emersonnii, Thermoascus aurantiacus, and Thermomyces lanuginosus) are in one way or another the prime fungal organisms that can degrade wood. The simple sugars generated in the hydrolysis step are then converted in the co-fermentation (second) step of the industrial process via thermophilic acetogenic bacteria into acetate, which, in turn, is converted to bio-methane and carbon dioxide by thermophilic methanogenic archaea. People may also note that thermophilic fungi are not known to have any special nutritional requirements as they can grow on simple media containing carbon, nitrogen, and mineral (K, Mg, Fe, Zn, and Cu) salts ^[16]. They are also mostly autotrophic. The ideal carbon (C) to nitrogen (N) growth ratio is twenty to one ^[16]. This is also the ideal C to N ratio for the fermentation step to optimize methane production, and it has been the basis of the selection of the respective amounts of wood wastes and manures in the “W2M3+2” industrial process, as indicated in the previous section ^[12]^[19]^[20]. It has been suggested that thermophilic fungi prefer non-ammonium-based nitrogen sources. Indeed, the supply of nitrogen in the nitrogen-rich manures is in its preponderance (70% to 80%) in a protein form, with the remainder being mineralized inorganic nitrogen in the form of ammonia. Another crucial, but usually overlooked, growth parameter of thermophilic fungi is the ratio of carbon (C) to phosphorous (P) ^[16]. The desired value of this ratio should be in the range of 120:1 to 240:1 to ensure optimal growth and is readily met in the industrial process as the nitrogen-rich manures are also phosphate-rich ^[12]^[19]^[20].

An important environmental factor of growth for thermophilic fungi is the pH factor. The pH of the environment exerts a profound influence on the transport of nutrients, solubility of nutrients, enzymatic reactions, and availability of specific metallic ions that may form soluble or insoluble complexes at a particular pH value. For example, metals like Mg, Fe, Ca, and Zn are available to a fungus at low pH values and become insoluble at higher pH values. Obviously, the pH preferences of individual fungi vary. Most thermophilic fungi tend to grow in the acidic range and/or a somewhat alkaline one, but they are also tolerant to a broad range of pH values from 4 to 8 ^[16]. These pH ranges are also consistent with the pH occurring in the hydrolysis step in the industrial process. As is to be expected, the pH in the hydrolysis step in the industrial process is highly dynamic in value and distribution. It varies across the reactor vessel from acidic to slightly alkaline and is affected by the biodegradation of the carbon and nitrogen sources. Optimal values of the pH are in the range of 5.5 to 8.9 for the fungal activities and from 6.0 to 7.5 for the bacterial activities ^[12]^[21].

The effect of oxygen on the growth of thermophilic fungi is obviously another critical environmental parameter, perhaps next to temperature in importance. The fermentation step of the industrial process is strictly anaerobic. However, in state-of-the-art dry digestion, and in anaerobic digestion systems such as ours, metered amounts of air are introduced into the digester such that the sulfides are oxidized to sulfates that remain in the liquid phase, thereby ensuring that the generated biogas is nearly free of hydrogen sulfide. The wood hydrolysis step where the thermophilic fungi constitute the primary microbial conversion agent varies from being aerobic to partially anaerobic across the reactor vessel. The consensus appears to be that fungi in general and thermophilic fungi more specifically are aerobic ^[16]^[17]^[21]. However, there is sufficient evidence to suggest that thermophilic fungi do not stop their growth in reduced oxygen environments, and in fact, they can survive well in anoxic conditions. For example, the pioneering investigation of the fungus Thermoascus aurantiacus indicated that its respiratory quotient (CO₂/O₂) remained practically the same, even at very low concentrations of oxygen, and that the fungus could withstand anaerobic environments continuously for 8 days without the loss of viability ^[22]. A study of the fungus Humicola insolens reported better growth under anaerobic or microaerobic conditions rather than aerobic ones ^[23]. Another study observed that the perfect (ascocarpic) condition of the fungus Penicilllium duponti vs. the fungus Talaromyces dupontii was initiated only under anaerobic conditions ^[9]^[17]^[24]. Studies on the requirements of oxygen in thermophilic fungi are limited, but it is believed that most of them require at least 0.2% oxygen for trace growth and 0.7–1.05% oxygen for moderate growth ^[16]. Obviously, the amount of oxygen in the hydrolysis step of the industrial process, “W2M3+2”, is a controlled parameter as it is in the fermentation step, albeit for entirely different reasons ^[3]^[4].

As it has been mentioned already in several instances, the wood hydrolysis step of the industrial process, while dominated by thermophilic fungi, is also aided by thermophilic actinomycetes. Actinomycetes are bacteria that grow in the form of mycelia, like that of fungi, with the difference being that bacterial mycelia have a typical dimeter of 1 mm vs. fungal mycelia, which have a typical diameter of 5 mm ^[25]. The name Actinomycetes is derived from the Greek words “aktin” for ray and “mykes” for fungus because of the morphological shape of these bacteria and their resemblance to fungi. It is also interesting to note that while the first antibiotic to ever be isolated was “Penicillin” from the mesophilic fungus Penicillium notatum in 1928 but did not go into production until 1942, two other antibiotics were isolated and crystalized from mesophilic actinomycetes: “Actinomycin” from Streptomyces antibioticus and “Streptomycin” from Streptomyces griseus in 1940 and 1942, respectively. Many more antibiotics have been derived from actinomycetes since then. Thermophilic actinomycetes consist of species in several genera such as Thermoactinomycetes, Thermomonospora, Microbispora, Sacharopolyspora, and Strepotmycetes ^[25]^[26]. For example, the thermophilic actinomycetes species, Strepotsporangium, is one of the many cellulose-degrading streptomyces ^[25]^[26]. Thermophilic actinomycetes are aerobic, Gram-positive, and have optimal growth temperatures of up to 75 °C, although they can survive at much higher temperatures ^[24]. While the aid of the thermophilic actinomycetes to thermophilic fungi in degrading cellulose has been known for more than eighty years, other thermophilic bacterial genera, i.e., non-actinomycetes, aiding in the process, directly or indirectly, have been identified in recent decades ^[11]^[27]^[28]. Thus, the thermophilic bacterium species, Thermus thermophilus, in the aerobic, Gram-negative genus, Thermus, grows in different piles of cellulosic and nitrogen-rich wastes (garden and kitchen wastes, sewage sludge) with optimal growth between 65 °C and 75 °C ^[25]^[27]. Moreover, a variety of bacteria that are homologous to the thermophilic bacteria species Bacillus schlegelii and Bacillus stearothermophilus in the Gram-negative genus Bacillus have been isolated in hot composts ^[25]^[28]. These thermophilic bacteria have an optimum growth at 70 °C to 75 °C, but under microaerophilic conditions of 5 kPa oxygen ^[28]. Undoubtedly, many more microbial species that have not yet been isolated and studied would exist in the wood hydrolysis step, because they can grow in that environment and consequently support the process in a symbiotic manner. Recent reviews on the biodegradation of wood, albeit in different environments than that of the “W2M3+2” process, confirm the extensive symbiosis of fungi with bacteria ^[29]^[30]. Forest ecosystems are estimated to store 861 Pg (Peta-grams) of carbon, of which about 73 Pg represent deadwood. Globally, wood decomposition into carbon dioxide is variously estimated to range from 2.1 to 11 Pg of carbon per year. The return of carbon dioxide into the atmosphere via wood decomposition is thus similar in magnitude to that from the current global fossil fuel combustion amounting to 9.5 Pg of carbon annually. (Note—1 Pg is equal to 1 billion metric tons.) It is interesting to note that the conversion of wood wastes globally per the “W2M3+2” process would reduce the emissions of CO₂ globally by an estimated amount of 2.6 billion metric tons of carbon per year just from not allowing the normal deadwood decomposition to occur and by thinning forests to reduce fires ^[4]. The estimated potential of waste wood to be collected annually to be processed into bio-methane is about 5.3 billion metric tons, of which about 2.6 billion tons is carbon, given that 48–50% of dry wood biomass is carbon by weight. The avoided carbon equivalent of annual greenhouse gas emissions from the utilization of pig manure and poultry litter to co-digest with the available waste wood is estimated to be 0.7 billion metric tons and 0.6 billion metric tons, respectively ^[31]. Thus, the application of the “W2M3+2” process technology would globally eliminate, at a minimum, some 3.9 billion metric tons of carbon annually. This is a quite significant amount, representing about 41% of the current fossil fuel carbon emissions of 9.5 billon metric tons per year ^[27]^[28]. It may take a period of about 20 to 30 years for the annual collection of most naturally occurring deadwood along with forest thinning to be established globally to sequester emissions of carbon dioxide and produce renewable natural gas. During the same time, the use of fossil fuels for power generation could be supplanted by the increased penetration of renewable wind and solar electricity, thereby curtailing the overall greenhouse gas emissions from fossil fuels. The global implementation of the “W2M3+2” process has the potential to supply over one-fourth of the current world energy use and 105% of the current world consumption of natural gas ^[4]. Assuming, for example, that this occurs by 2050, and at the same time, some 50% of electricity production globally is based on wind and solar power, then the fossil fuel carbon footprint would be diminished to at least one-half and likely to one-third its present value, given that global power generation relies heavily on coal combustion and that “W2M3+2” bio-methane production can essentially replace most if not all of fossil and natural gas use. Thus, the global implementation of the “W2M3+2” process could, by 2050, totally offset the emissions from fossil fuels at that time and ultimately lead to a net carbon dioxide removal from the atmosphere.

Based on our knowledge of the anaerobic co-digestion of organic wastes as well as our current understanding of the wood to bio-methane conversion in lower termites, it is to be expected that the industrial wood hydrolysis step in the “W2M3+2” process relies on a multitude of thermophilic fungi that operate synergistically with certain bacteria and perhaps even other microorganisms to be identified over time.

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1. Introduction

2. The Thermophilic Fungi of Choice for Bio-Methane Production

References