Professors from the Tokyo Institute of Technology present their latest work in various biotechnological fields, with applications in sustainable manufacturing, biofuel production, agriculture, and material science.
by Oh Sher Li
The 2020 Tokyo Tech Research Showcase was held in September, with the theme “Biotechnology for Industrial Use”. Organised by Tokyo Tech ANNEX Bangkok, the showcase was co-sponsored by Tokyo Institute of Technology and the National Science and Technology Development Agency (NSTDA) of Thailand, with the support of the Tokyo Tech Alumni Association.
The virtual event featured recent and current research in biotechnology with agricultural applications. These studies ranged across various disciplines from microbiology to chemistry, and their applications extend from waste management to fuel production and even synthetic materials. In this article, we look at each of these studies, and explore their potential uses in agriculture and sustainability.
Microbial synthesis of biodegradable polyesters
The generation of plastic waste is a major environmental concern, stemming from large amounts of plastic being produced for various industries, which ends up being incinerated, accumulating in landfills, or in the ocean as harmful microplastics. To alleviate the problem of plastics polluting the environment, the use of traditional petroleum-based plastics by industries could be replaced by the adoption of biodegradable thermoplastics.
Professor Toshiaki Fukui from the School of Life Science and Technology at the Tokyo Institute of Technology has developed a type of biodegradable polyester known as polyhydroxyalkanoates (PHAs) from Ralstonia eutropha bacteria. According to Professor Fukui, this biodegradable plastic can be used in the same applications as regular plastics but has the added advantage of being environmentally friendly, as it can be degraded to carbon dioxide and water by microbial organisms.
PHAs are naturally occurring polyesters produced by microorganisms such as bacteria. Poly[(R)-3-hydroxybutyrate] [P(3HB)] is one of the most abundant PHAs found in nature, although its homopolymer is brittle due to its highly crystalline structure. P(3HB) is produced by R. eutropha bacteria and can then by extracted and processed to produce biodegradable plastics.
In order to enhance the flexibility of the PHA product, Professor Fukui’s team developed a bio-polyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)]. This copolymer has a lower melting temperature, lower crystallinity, and can undergo much higher elongation before breaking, as compared to the homopolymer of P(3HB). Furthermore, P(3HB-co-3HHx) remains highly biodegradable even in marine environments, making it an attractive alternative to traditional polymers which break down into microplastics and contribute to water pollution.
The biosynthesis of P(3HB-co-3HHx) was achieved by genetically engineering R. eutropha, so that the bacteria could make use of renewable sources such as vegetable oils for copolymer synthesis, in the presence of carbon dioxide and hydrogen as carbon and energy sources.
Professor Fukui hopes that the development of biodegradable polyesters that are produced from renewable resources could provide sustainable alternatives to manufacturing industries.
Producing biofuel from microalgae
In order to reduce our dependency on fossil fuels, we need to research and develop alternative sources of energy. Such renewable sources of energy may include solar, geothermal and wind power. However, for many industrial uses and personal vehicles, liquid fuel such as gasoline and diesel remain most practical and affordable, which will likely hold true even in the future. Therefore, another aspect in sustainable fuel production and consumption that could be of interest is the reduction and minimisation of carbon dioxide emissions from “well to wheel”, or from fuel extraction and production to consumption.
Professor Hiroyuki Ohta’s research group from the School of Life Science and Technology at Tokyo Institute of Technology has developed a method of producing biofuel from microalgae, by utilising lipid remodelling under nutrient starvation. Specifically, nitrogen and phosphorous starvation was used to induce oil accumulation in Chlamydomonas reinhardtii, which is a single-cell green alga.
Lipid remodelling is often observed in microalgae in response to limited availability of nitrogen and phosphorous. This phenomenon is characterised by enhanced synthesis and accumulation of triacylglycerides, which are a type of fatty acids used in biodiesel production.
In his research, Professor Ohta observed that phosphorus starvation induced lipid remodelling and oil accumulation in C. reinhardtii, and that this behaviour was conserved in both algae and land plants. In addition, it is suggested that betaine lipids and sulfolipids substitute the phospholipids in the extraplastidic membranes of microalgae during phosphorous deficiency in the microalgae, and that membrane remodelling in the chloroplasts promotes the production of sulfoquinovosyl diacylglycerol (SQDG) rather than the usual phosphatidylglycerol.
Professor Ohta’s team discovered that the regulatory mechanism leading to lipid remodelling under conditions of phosphorus starvation is conserved in both primary and secondary endosymbionts. Endosymbiosis refers to a form of symbiotic relationship, in which the endosymbiont lives in the body of another organism, usually in a mutualistic relationship. Professor Ohta and his team sought to investigate the reasons behind why lipid remodelling is such a highly conserved mechanism, what exactly is conserved in this regulatory mechanism, and how the process is regulated in organisms.
There are a few known major regulatory factors for phosphate starvation, namely PSR1 in C. reinhardtii and PHR1 in Arabidopsis thaliana. By using the Algae Gene Coexpression Database (ALCOdb), Professor Ohta’s team looked at genes that were co-expressed with diacylglycerol acyltransferase (DGAT) genes, which are involved in TAG synthesis, as well as transcription factors that were co-expressed with DGTT1, which is a DGAT gene whose expression is upregulated under conditions of nitrogen and phosphorus starvation.
From their investigations, the researchers found that in C. reinhardtii, lipid remodelling regulator 1 (LRL1) and DGTT1 had similar expression patterns. They further explored the role of LRL1 and found that reduced expression resulted in repressed lipid accumulation and SQDG synthesis in phosphorus starvation. Professor Ohta and his team hypothesise that the PSR1 and LRL1 proteins activate phosphorus starvation-induced gene expression in early phase and late phase respectively in C. reinhardtii.
Besides identifying genes involved in lipid remodelling under phosphorus starvation and their regulatory roles, Professor Ohta and his team also studied lipid compartmentation in Nannochloropsis oceanica. Namely, omega-3 fatty acids accumulate in the chloroplast and facilitate the efficient utilisation of lipids, while saturated fatty acids used in biodiesel production accumulate in oil droplets within Nannochloropsis cells.
Enhanced utilisation of resources and waste for the environment
Biotechnology has the potential to solve environmental issues. Professor Kiyohiko Nakasaki from the School of Environment and Society at Tokyo Institute of Technology and his team aim to solve environmental issues through microbial activities, to reduce waste production and recover energy and material resources.
The research endeavours presented by Professor Nakasaki are centred on DNA information-based engineering, which is a method used to control the quality of biological products and processes based on genetic information. Specifically, his research team used molecular biology techniques to investigate the optimal compost maturity that could best promote plant growth.
Compost is made from the degradation of organic matter and differs from regular potting soil since it contains a high level of nutrients that promote plant growth. The process of compost formation involves several microorganisms, which exist in a dynamic microbial consortium which changes as organic matter degradation progresses. The degradation can be measured by determining how much carbon has been converted to carbon dioxide in relation to the total amount of carbon present in the raw material.
Using these principles, Professor Nakasaki and his team carried out a plant growth assay investigating the performance of compost at various stages of organic matter degradation, in comparison with conventional chemical fertilisers. From these experiments, they concluded that compost that had undergone 40 to 70 percent conversion of total carbon yielded the best results in terms of promoting plant growth.
Professor Nakasaki’s team also evaluated the safety of the compost at various stages of organic matter degradation, to determine if the compost would be suitable for agricultural purposes, particularly for edible crops. They found white fungi on the soil surface of plants grown with compost that had undergone 40 percent or less of carbon conversion. This implies that compost with less than 40 percent organic matter degradation is potentially susceptible to pathogenic fungi and may not be safe for agricultural purposes.
To examine the microbial consortium of compost at varying levels of organic matter degradation, the researchers used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). This experimental technique allows large DNA fragments of similar sizes to be separated based on DNA sequence mutations, which are unknown at the time of the experiment. By employing PCR-DGGE and subsequent gene sequencing and phylogenetic studies, Professor Nakasaki’s laboratory obtained data suggesting that a shift in the microbial consortium, specifically a reduction in Bacillus pallidus bacteria and increased presence of Bacillus thermoglucosidasius bacteria, is responsible for the improved quality of the compost in promoting plant growth as the compost matures.
Since it can be inefficient to depend solely on plant growth assays to determine the quality of each batch of compost, particularly in large-scale commercial settings, Professor Nakasaki’s team proposed a method of screening for compost quality based on their findings. They designed a specific oligonucleotide for the detection of B. pallidus, which undergoes hybridisation with antibodies and produces a blue colour when reacted with a substrate solution. The blue colour serves as a reporter for the presence of B. pallidus, allowing the researchers to determine the point when the bacteria is reduced and indicating that the compost could be ready for use.
If successful, the assay developed by Professor Nakasaki and his team could be a much faster and simpler way to predict the effectiveness of compost in promoting plant growth, in contrast with traditional assays which observe plant growth after introducing compost for several weeks. Professor Nakasaki hopes that this work could improve quality control workflows and be integrated into agricultural practices, to streamline processes and boost production of agricultural products.
Sugar conversion to monomers for high performance polyamides
High performance polyamides are a type of thermoplastic synthetic resin with high melting points, glass transition temperature (the temperature at which the material changes from a brittle state to a more flexible, rubbery state), chemical resistance and stiffness. They have several important industrial applications, including in medical catheters, electrical connectors and in pipes and supply lines for oil.
The production of high-performance polyamides is difficult and costly. Professor Michikazu Hara from the Institute of Innovative Research at Tokyo Institute of Technology conducts research on how to convert biomass, specifically glucose, to high performance polyamides. While other studies claim to have been successful in doing this, these methods are still rather inefficient and expensive, and can be improved upon greatly.
The first step to doing this is the conversion of glucose to 5-(hydroxymethyl)furfural (HMF). This involves a hydride transfer converting glucose to fructose, then intramolecular dehydration, or the removal of water, to obtain HMF. Professor Hara’s team identified phosphorylated titanium dioxide, P-TiO2, as a potential catalyst. They investigated various setups and were eventually able to achieve an isolated HMF yield of 94 to 96 percent from a glucose solution, with over 99 percent purity as assessed by nuclear magnetic resonance (NMR) spectroscopy.
The next step in the synthesis of high-performance polyamides is the conversion of HMF to 2,5-Bis(aminomethyl)furan (AMF). To do this, the team carried out a first reductive amination of HMF to aminomethyl hydroxymethyl furan (AHF), then AHF was converted to AMF by borrowing hydrogen using a heterogeneous catalyst. The methods developed in this investigation for both of these steps improve upon older methods used previously and give high AMF yields.
Besides the scalability and high yields of the techniques developed and studied by Professor Hara’s lab, these methods provide another important advantage making them more attractive for industrial adoption — they could greatly reduce the cost of production of high-performance polyamides. Using these methods, the production cost of HMF is reduced to less than USD 1 per kilogram, and that of AMF is less than USD 5 per kilogram, in part because they use glucose from biomass as raw material for production.
The studies presented by the speakers at the 2020 Tokyo Tech Research Showcase demonstrate the wide range of fields that can be studied using biotechnology, and how there are still many ways that biotechnology research can contribute to agricultural and industrial applications. Several of these studies even go beyond industrial needs and targets, by focusing on minimising negative environmental impacts of current practices while maximising yield. These are just four of the many ongoing projects around the world seeking to improve manufacturing techniques by increasing yield and efficiency, all while reducing cost and environmental damage, and give a glimpse into what the future could hold for several industries.
- Denaturing Gradient Gel Electrophoresis | Springer Nature Experiments. (2017). Retrieved October 24, 2020, from Springernature.com website: https://experiments.springernature.com/articles/10.1385/1-59259-273-2:125