Holistic biology could pave the way for Japanese biofuels

Insights gained by the Biosphere Oriented Biology Research Unit into complex biological systems could revolutionize energy production and re-integrate society into the global ecological cycle

Figure 1: Carbon cycle of the ecological system. Carbon circulates through the global ecological system. Coal and petroleum are naturally isolated from the carbon cycle, and the consumption of these fuels on a global scale is causing environmental problems such as environmental pollution and global warming

Author:

Shigeharu Moriya
Research Unit Leader
Biosphere Oriented Biology Research Unit
RIKEN Advanced Science Institute

“Japan is very rich in forests and living marine resources,” says Shigeharu Moriya, research unit leader of the Biosphere Oriented Biology Research Unit at the RIKEN Advanced Science Institute. Forests cover about one-third of Japan’s land area, and the country’s marine boundaries including the exclusive economic zone and territorial waters takes in the tenth largest area of sea in the world. “If we can take advantage of these resources to produce biofuels efficiently, Japan could become an energy-exporting country.” Moriya’s research unit is striving to develop an efficient approach for producing biofuels by studying biological activities as a whole, including the activities of protist microorganisms that live in the gut of termites and degrade the cellulose in wood fiber, as well as the activities of groups of microorganisms that contain oil-producing algae.

Re-integrating human society into the global ecological system

“Human society was integrated into the global carbon cycle of the ecological system before the Industrial Revolution in the eighteenth and nineteenth centuries,” says Moriya. Plants take in carbon dioxide to synthesize organic compounds through photosynthesis. Animals eat those organic compounds, and their remains and waste are in turn degraded into inorganic substances by microorganisms such as bacteria and algae. Animals, plants and microorganisms provide carbon dioxide through respiration, and the carbon dioxide is again used by plants during photosynthesis. In this way, carbon circulates through the ecological system (Fig. 1).

“After the Industrial Revolution, however, human society began to consume huge amounts of fossil fuels such as coal and oil, which had been left in the ground and isolated from the carbon cycle. It is the huge consumption of fossil fuels that has caused the current problems of environmental pollution and global warming due to carbon dioxide emissions.” Human society is also facing the serious problem of the depletion of fossil fuels. “We can take advantage of science and technology to integrate human society into the carbon cycle of the ecological system again by using renewable biological resources such as plants efficiently to produce biofuels and bioplastic materials. If this integration is achieved, we will be able to establish a sustainable society.”

Using unique approaches to probe the functions of microorganisms

In the natural world, biological resources are used efficiently to produce various useful substances. “Microorganisms take the leading role. We just do not know how they produce useful substances efficiently. We should learn their approach.”

Biofuels can already be produced by converting the starch contained in cereal grains such as corn and sugarcane into sugars, which are then fermented into biofuels. This method, however, is inefficient, consuming large amounts of energy, competing with the production of food and restricting the use of agricultural land. Researchers all over the world are now striving to produce biofuels from the cellulose contained in wood chips, wood thinned from forests, rice straw, and even weeds. Existing fermentation technology can be used to produce alcohol efficiently provided that cellulose is degraded into glucose, a kind of sugar. The point is that cellulose is very difficult to degrade.

“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.”

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. 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.”

There are several to a dozen types of protists in the intestines of termites (Fig. 2). 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.”

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. 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. “We kept an eye on mRNA, proteins and metabolic products that are actually working. To begin with, we examined the mRNA of the protists that live in various kinds of termites because we 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 (Fig. 3). “Normally, even 1% is considered to be a large expression level for gene groups related to a single function. Thus, 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. “Fungi such as T. reesei also use the same types of cellulase. When we compared fungi and protists in terms of cellulase, we found that the cellulase produced by protists can degrade cellulose into glucose at least ten times more efficiently than that produced by fungi. We 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.”

New mechanisms hidden in unknown genes

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, 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. We believe that the new lignin-degrading mechanism is hidden in those genes.”

Those unknown genes (Fig. 3) must produce proteins. “We moved termites from an environment where they are raised on wood to an environment where they are raised only on cellulose. We found an increase in the amount of glucose produced by protists. We are 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. “If glucose is produced, we 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 study to include the sea. “In the sea, certain algae use photosynthesis to produce oil. We try to expose ourselves to the habitat of our target organisms before starting our research. Thus, before starting our research into algae, we performed a dive in the Odaiba area of Tokyo Bay. The water was so cloudy just five meters below the surface that we needed a flashlight. The sea was thick with microorganisms such as algae because of eutrophication.”

Many algae have a calcareous or glassy shell. “The shell is heavy enough to cause them to sink to the sea bottom, preventing photosynthesis. Thus, 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.”

Researchers are now competing to develop an algae-based technique to produce biofuels. “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.”

Moriya is focusing on activities in which multiple organisms exchange substances between them, rather than on a single group of organisms. “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.” 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. “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.”

The outlook, however, is far from set in stone. “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.”

A new field of holistic biology

Moriya has been interested in strange living organisms since he was a child. It was 1996 when he started research on the protists that live in the intestines of termites. “I took a post in Thailand for about a year from 2000 because the laboratory I belonged to started a joint research with a laboratory there. In Thailand I was overwhelmed by the diversity of living things in the tropical region, where a complex, symbiotic network is established. Through that experience, I realized that what I needed was to study biological activities as a whole rather than to study the activities of individual organisms isolated from their symbiotic network. After returning home, I started studying mRNA in the symbiotic condition rather than isolating protists from termites.”

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. “I named the approach that studies biological activities such as genes, mRNA, proteins and metabolic products without separating them from their ecological system ‘metaomics analysis’. RIKEN is equipped with advanced facilities and the human resources necessary to conduct this type of analysis. I want to take advantage of these research facilities to establish the technology for metaomics analysis as well as explore a new field of science that can help in studying the complex ecological system as a whole.”

Moriya believes that developing an understanding of the mechanism of the ecological system and creating original techniques to produce biofuels are two sides of the same research. “They cannot be separated. Using science and technology to re-integrate human society into the carbon cycle of the ecological system can only be achieved by understanding the essence of the ecological system itself.”

About the Researcher

Shigeharu Moriya

Shigeharu Moriya was born in Tokyo, Japan, in 1969. He obtained his PhD degree from the graduate school of Yokohama City University in 1996. After three years postdoctoral training at RIKEN, he became a researcher at RIKEN. From 1999 to 2001, he worked at the Kasetsert University of Thailand as a Japan Science and Technology Agency researcher. He continues his research activity at RIKEN after returning from Thailand and also became a guest researcher of Yokohama City University. He has been senior researcher and unit leader of the Biosphere Oriented Research Unit since 2008, and guest associate professor of Yokohama City University since 2010. His research focuses on the whole flow and interaction of organic compounds and environmental microbes in the complex ecological system, including the symbiotic systems of higher organisms.

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Figure 2: 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

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Figure 3: Analysis of genes that are active in groups of protists 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