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RIKEN team taking holistic biology approach to develop more efficient methods for producing biofuels; protists in termite guts and marine algae

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Japanese subterranean termites and groups of protists that live in their intestines. A dozen types of protist live in the intestines of Japanese subterranean termites, the most common type of termite in Japan. Source: RIKEN. Click to enlarge.

The Biosphere Oriented Biology Research Unit at the RIKEN Advanced Science Institute in Japan is striving to develop efficient approaches for producing biofuels by studying biological activities as a whole. Two prime areas of focus for the group are the activities of protists (a diverse group of eukaryotic microorganisms) that live in the gut of termites and degrade the cellulose in wood fiber; and the activities of groups of microorganisms that contain oil-producing algae.

First-generation biofuels can already be produced by converting the starch contained in cereal grains such as corn or sugarcane into sugars, which are then fermented into biofuels. This method, however, is inefficient on a number of fronts, notes Shigeharu Moriya, Research Unit Leader. Existing fermentation technology can be used efficiently to produce second-generation biofuels from cellulose, provided that cellulose is first degraded into glucose—and cellulose is very difficult to degrade, Moriya adds.

Cellulose has so far been degraded mainly using Trichoderma reesei, which is a fungus that is capable of degrading cellulose into glucose because it produces large amounts of cellulase, a cellulose-degrading enzyme.

—Shigeharu Moriya

However, before using T. reesei to degrade cellulose, it is first necessary to remove the lignins that entwine the cellulose. Because it is very difficult to remove lignin using existing enzymes, this process requires preprocessing with sulfuric acid at high temperatures, which in turn requires preprocessing and treatment of waste liquids and the consumption of large amounts of energy, Moriya says. For practical application, it will be necessary to reduce the energy consumption as much as possible.

In the natural world, certain organisms proliferate because of their ability to degrade cellulose more efficiently. For example, termites live only on wood. The cellulose contained in the wood is degraded by protist organisms that live in the termites’ intestines. However, we do not know the mechanism of how they degrade the cellulose. Our unit is aiming to produce biofuels efficiently by taking advantage of this mechanism.

—Shigeharu Moriya

There are several to a dozen types of protists in the intestines of termites. The protists are also host to various bacteria. Termites, protists and bacteria exchange the substances essential for their survival, establishing a codependent relationship.

Because of that codependent relationship, we have not been able to isolate and cultivate the protists to study their characteristics and functions.

—Shigeharu Moriya

“Microorganisms take the leading role [to produce various useful substances]. We just do not know how they produce useful substances efficiently. We should learn their approach.”
—Shigeharu Moriya

Separation and culture techniques for the purpose of studying microorganisms have been developed over many years. Current techniques, however, can only be used to cultivate less than 1% of all microorganisms. Moriya says. The characteristics and functions of most microorganisms therefore remain unknown. A recently developed technique called metagenome analysis allows the genomes of multiple groups of organisms as a whole to be analyzed without cultivation. This approach, however, can only provide a list of genes; it provides almost no information on which genes are working and how they fulfill their specific functions.

Genetic information is encoded as a linear sequence of four bases that make up the DNA. The base sequence of a gene region, which is part of the DNA, is transcribed as messenger RNA (mRNA), and the information is used as a base for the production of enzymes and proteins. The proteins then work to produce metabolic products.

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Measurements of mRNA expression in the protists that live inside various kinds of termites have revealed that 5–10% of mRNA expression is associated with genes (red) related to cellulose-degrading enzymes. Approximately 50% of mRNA expression was found to be associated with genes (dark blue) with unknown functions. Source: RIKEN. Click to enlarge.

The RIKEN team began by examining the mRNA of the protists that live in various kinds of termites because it thought that analyzing the base sequence of this mRNA would provide information on which genes are be expressed and by how much.

Moriya’s experiments showed that enzymes related to cellulose degradation accounted for 5–10% of all expression. Given that even 1% is considered to be a large expression level for gene groups related to a single function, Moriya says, the team concluded that it is safe to say that the protists in termites live only to degrade cellulose.

The experiments also revealed that cellulase, a cellulose-degrading enzyme, comes in five different types. When the RIKEN team compared fungi and protists in terms of cellulase, its found that the cellulase produced by protists can degrade cellulose into glucose at least ten times more efficiently than that produced by fungi. It also found that the cellulase has “mysterious characteristics.”

The cellulase produced by fungi has a cellulose-binding site. If the binding site is removed artificially, the activity of cellulase is dramatically reduced. The cellulase is considered to exhibit strong activity when it is securely bound to cellulose. In contrast, the cellulase produced by protists has no binding sites at all, yet its activity is at least ten times higher.

This mystery has yet to be clarified. In collaboration with researchers at the RIKEN SPring-8 Center, we are now advancing our research by crystallizing cellulase produced by Protists and examining its structure using X-rays. We think that a structural comparison between the cellulase produced by fungi and protists will allow this mystery to be resolved. So far, we have not yet discovered any enzymes like this cellulase that have no binding sites and yet exhibit strong activity. We may be able to create an enzyme with strong activity if the mechanism is clarified at the structural level.

—Shigeharu Moriya

Fungi use enzymes to produce active oxygen, which is then used to degrade lignin. Active oxygen, however, cannot be produced in the intestines of the termites in which the protists live because there is no oxygen. Thus, Moriya says, protists must have a completely different lignin-degrading mechanism. The analysis of the mRNA expression in protists showed that half of the expression is related to unknown genes. The team believes that the new lignin-degrading mechanism is hidden in those genes.

The team then moved termites from an environment where they are raised on wood to an environment where they are raised only on cellulose; the researchers found an increase in the amount of glucose produced by protists. The team is now studying what types of proteins are produced in this cellulose-only environment; the proteins could include the unknown enzymes that are related to lignin degradation.

According to Moriya’s estimates, it could be possible to improve the energy efficiency of biofuel production from cereal grains such as sugarcane by as much as 2.7 times if enzymes are used to remove lignin and the cellulase used to degrade the cellulose is at least ten times stronger than that produced by T. reesei.

Furthermore, if glucose is produced, they will be able to produce bioplastics in addition to biofuels. In April 2010, RIKEN established the RIKEN Biomass Engineering Program (BMEP) with the aim of creating new materials such as bioplastics. Moriya and his unit members are also advancing their research in collaboration with BMEP.

Producing fuels from marine algae. Moriya and the members of his research unit have extended their studies to include marine algae. Many algae have a calcareous or glassy shell; the shell is heavy enough to cause them to sink to the sea bottom, preventing photosynthesis. As a result, says Moriya, they produce oil for buoyancy.

Microorganisms such as algae produce oil from phosphorus and nitrogen that they absorb from their environment. As phosphorus and nitrogen are causes of marine pollution, if we can take advantage of the function of these microorganisms, we will be able to produce biofuels while purifying the sea. Most researchers, however, have focused on a single group of algae, such as Chlamydomonas in the United States and Botryococcus discovered by Prof. Makoto Watanabe at Tsukuba University.

—Shigeharu Moriya

Moriya is focusing on activities in which multiple organisms exchange substances between them, rather than on a single group of organisms. Moriya’s research unit has already have begun experiments using seawater samples containing groups of microorganisms including algae collected from Tokyo Bay and from the inner bay of Iriomote Island

We do this because exchange is one of the main activities among organisms in the natural world. I believe that there is an efficient oil-synthesis pathway that can be achieved only when multiple living organisms exchange substances between them.

As the darkness increases, the algae will produce more oil so that they can float to the surface to receive more light. Thus, we are planning to build an experimental setup that can stimulate algae to produce more oil, to monitor the proteins or metabolic products produced by groups of microorganisms, and to analyze how organisms exchange substances between them. We aim to use the results to discover an efficient oil-synthesis pathway.

Our destination is still unclear because we are just at the earliest stage of our research. Under the sea, light can reach a depth of 30 meters, and surface waves have little effect on the movement of seawater. One researcher has proposed the idea of growing algae within an undersea region that is free from typhoons and surrounded by a translucent membrane. If we can discover further efficient oil-synthesis pathways, we will be able to produce huge amounts of biofuels using the sea areas around the Japanese islands. Thus, if we can take advantage of forests and living marine resources to produce biofuels, Japan could become an energy-exporting country.

—Shigeharu Moriya

The Biosphere Oriented Biology Research Unit lead by Moriya has been conducting research in close collaboration with the Advanced Nuclear Magnetic Resonance Metabolomics Team headed by Jun Kikuchi at the RIKEN Plant Science Center.

Comments

joewilder

If there was ever anything that could convert wood in excrement, it's termites. Take it from me - I lived in Hawaii for a few years. Termites can eat a building right behind you as you're erecting it.

kelly

Whatever works.

SJC

They could come up with a better understanding and new methods along this path. I would just gasify and synthesize whatever fuels are needed.

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