Fungi Paving the Way into a New Biofuel Based Future

With a growing need to develop alternative, cleaner methods for producing fuels, attention has been turned to the production of biofuels. The production of second generation biofuels is more sustainable than the production of first generation biofuels because not only does it reduce the need to use food crops, but it uses a higher number of substrates, thus maintaining a higher biodiversity of plants being used. Even though the use of second generation biofuels is more sustainable, the cost to produce such fuels currently is a limiting factor in the production because there are many barriers preventing cost-effective production. One such limitation includes finding enzymes that are suitable for use in industrial settings: the enzymes must be rapid, cheap and thermotolerant [3]. Currently, the industrial process for biomass degradation for biofuel production is incredibly expensive and quite slow, while the conditions for degradation are acidic and hot [4]. In a recent study, the genomes of two thermophilic fungi have been sequenced: Thielavia terrestris and Myceliophthora thermophila [1]. A closer look will be taken at M. thermophila. Because these are the first thermophilic, filamentous fungi to have genomes sequenced, insight can be gained into what a fungi needs at the genomic level to be useful in biomass degradation and biofuel production.

First and second generation biofuels: the difference.

Figure 1. The production of biofuels from Rubin et. al. Solar
energy is stored in plant cells in various compounds that can be
broken apart with chemicals. The smaller components (simple
sugars) can then be used by microbes to produce biofuels. [4]

Before discussing the why M. thermophila might be useful in the production of second generation biofuels, it is important to establish what first and second generation biofuels are. A look at Figure 1 will give a brief overview at how biofuels are produced. First generation biofuels are typically made from food crops and can be mixed into petroleum based fuels. Examples of these include bio-ethanol and bio-esters (biodiesel). A problem is posed that the production of 1^st generation biofuels require the use of food sources, so the price of food stocks go up while the availability becomes more limited. In the production of second generation biofuels, plant biomass not derived from food stocks are used. An example of a second generation biofuel that might be familiar is cellulosic ethanol. In 2^nd generation biofuels, hardy enzymes that come from tough microbes are a requirement. Without these, there breakdown of the components in plant cell walls would not occur at a fast rate. Second generation biofuels avoid food sources and are thought to be able to contribute significantly to the reduction of CO₂ production [3]. Though these biofuels can be thought of as the next up and coming way to make fuels, certain organisms must be used in the degradation process because not all microbes have what it takes to be part of biomass degradation.

What’s so special about M. thermophila?

Figure 2. M. thermophila [2]

M. thermophila (Figure 2) is a fungi ubiquitously found in compost piles. The compost heaps these fungi call home reach temperatures that prevent the growth of many other microbes, yet these fungi maintain a love for the heat. The enzymes of M. thermophila function better at temperatures above 45°C than they do at 25°C. Not only do these enzymes function at high temperatures, but in relation to commonly used species in degradation, the thermotolerant enzymes have a higher hydrolytic capacity: they are more efficient, so less are required to complete the same amount of degradation. Currently the most widely used cellulases in biomass degradation function optimally between 40°C and 50°C but are incredibly slow. In order to increase enzymatic activity and reduce contamination at large scale, the temperature of the reactor can be ramped up; however, with non thermophilic fungi used in the process, the enzymes of these fungi can be denatured and rendered useless. Recent genomic analyses as well as a look into the optimal functioning temperature of enzymes of M. thermophila have provided insight into these fungi contain which make them special when it comes to biodegradation [1].

Thriving in the heat.

Table 1. Optimal temperature of four different xylanases in M. thermophila extracted from Berka, et. al 2011 [1].
Gene ID	Family	Optima (°C)
Mycth_100068	GH11	50
Mycth_2121801	GH11	60
Mycth_112050	GH10	60
Mycth_116553	GH10	70

M. thermophila is a thermophilic fungi, characterized by its ability to grow better at temperatures above 45°C than at 25°C. Some of the enzymes harbored by this thermophile function optimally at temperatures upwards of 70°C. In the Berka, et. al study, the hydrolysis of alfalfa by numerous enzymes was examined at various temperatures to determine the optimal functioning temperature of M. thermophila. Xylanases, enzymes that break down hemicellulose (a component of plant cell walls) show a wide range of optimal temperature functioning. After inserting the genes for four different xylanases into Aspergillus niger, it was shown that different enzymes have different optimal temperatures at which they function, as can be seen in Table 1. By having enzymes that function at different temperatures, M. thermophila is ensuring that it is able to degrade biomass at a wide range of temperatures [1]. Think about it: compost heaps, this  organism’s niche, are not always at a consistent temperature, so by being able to adjust to changes, M. thermophila is increasing its chances of survival. Not only is it important to look at enzymatic function, but it is important to go smaller and take a look at what makes up these proteins.

It’s the small differences that count.

It is important to look at the differences between well studied thermophiles and this newly sequenced organism. Most well studied thermophiles that have been sequenced are prokaryotes. The Berka, et. al study looked at the nucleotide composition of M. thermophila in comparison to 6279 different orthogroups. In prokaryotes, it has been shown that GC content has nothing to do with making the microbe a thermophile. However, it was found that 75% of functional genes have a higher GC content at the third position of a codon in M. thermophila. This number is significantly higher than other mesophiles studied. In regards to amino acid composition of thermophilic prokaryotes, a trend is seen in which there is a higher ratio of glutamic acid, arginine and lysine while they have lower than average alanine, aspartic acid, glutamine and asparagine composition. This trend was not carried over into M. thermophila: the amino acid content of the proteins analyzed showed no significant difference in composition to non-thermophilic fungi, such as Saccharomyces species [1]. M. thermophila may not have many things in common with thermophilic prokaryotes at the genomic and proteomic level, but both inhabit high temperature environments. Having properties that allow this organism to thrive in the heat will be beneficial in biofuel production because of the high temperatures required in the degradation processes.

The all-purpose decomposers.

The carbohydrate active enzymes (CAZymes) of M. thermophila were compared to the CAZymes of 8 other fungi and genomic evidence shows that M. thermophila is an all-purpose decomposer in the degradation of plant polysaccharides. It was found that M. thermophila harbored a large number (more than 200) of glycoside hydrolases and polysaccharide lyases, a common occurrence in thermophilic fungi. M. thermophila grows and decomposes better in neutral to alkaline environments. This is due to the fungi harboring a large number of pectin hydrolases, enzymes that function better at neutral pH. Looking at GH61 family of proteins (enzymes that degrade plant wall material) provides further insight into the diversity of the enzymes held by these fungi: there are 25 very different orthologs of GH61. Not only are there many orthologs, but in the presence of various metals, they have the ability to increase the hydrolytic activity of cellulases rapidly while reducing the amount of these cellulases needed for the degradation [1]. Recall that fermentation units need to contain rapid enzymes while working at fairly acidic conditions. Even though M. thermophila prefers neutral conditions, it is still able to grow in some acidic conditions. With the knowledge of productivity and hardiness of the M. thermophila enzymes, it is looking promising to use these fungi in the production of biofuels. M. thermophila has many qualities that make it an ideal organism to use in the breakdown of plant mass for the production of second generation biofuels. With the study of thermophilic fungi, such as this one, barriers to cost-effective production of biofuels can start coming down.

Submitted by Michelle Goettge.

Resources

1.      Berka, Randy M; Grigoriev, Igor V; Otillar, Robert; Salamov, Asaf; Grimwood, Jane; Reid, Ian; Ishmael, Nadeeza; John, Tricia; Darmond, Corinne; Moisan, Marie-Claude; Henrissat, Bernard; Coutinho, Pedro M; Lombard, Vincent; Natvig, Donald O; Lindquist, Erika; Schmutz, Jeremy; Lucas, Susan; Harris, Paul; Powlowski, Justin; Bellemare, Annie. (2011) Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nature Biotechnology. 29(10): 922-927.

2.     DOE Joint Genome Institute. (2011) Advancing Next Gen Biofuels by Turning Up
the Heat on Biomass Pretreatment Processes. http://jgi.doe.gov/News/news_11_10_02.html

3.     Naik, S.; Goud, V.; Rout, K and Dalai, A. (2009) Production of first and second generation biofuels: A comprehensive review. Renewable and Sustainable Energy Reviews 14 (2010): 578–597.

4.     Rubin, Edward. (2008) Genomics of cellulosic biofuels. Nature: Reviews. 454: 841-845.