Wastewater containing high concentrations of oils and fats is a serious problem because it causes not only pollution problems such as eutrophication in aquatic environments but also malfunction of wastewater treatment facilities such as conventional activated sludge process, membrane bioreactor, or anaerobic digestion process. Conventionally, pressure floatation equipment has been used, while it brings industrial waste of sludge and gives off offensive odors. Microbial degradation has been studied as alternative methods. However, the oil degradation ability of microorganisms demonstrated was too low to substitute the pressure floatation equipment.
We developed a symbiotic microbial agent which consists of three types of microorganisms isolated by our group, and succeeded in dramatically accelerating the oil-degradation rate. The mechanism is as follows: A bacterium, Burkholderia arboris, secrets lipase and hydrolyzes oils and fats (triacylglycerol) into fatty acids and glycerol and degrades the fatty acids. Then, two kinds of yeasts, Yarrowia lipolytica and Candida cylindracea, degrade the fatty acids and glycerol, respectively. By using this symbiotic relationship, oils and fats can be converted into inorganic carbons or microbial cells without reverse reaction which is attributed to the accumulation of the fatty acids and glycerol. This microbial agent showed remarkable oil degrading capacity compared with other existing oil degrading microbial agents. This high oil degradation ability allows us to substitute pressure floatation equipment with our microorganism agents. By installing our products, decrease in offensive odors from sludge, reduction of industrial waste, simplifying the treatment process, and space-saving would be achieved, resulting in a cost-effective pretreatment system for oils and fats in wastewater.
3rd generation biorefinery research utilizing microalgae is ongoing in many countries because fuel/chemicals production from carbon dioxide by photogenic microorganisms is essentially sustainable. Among various photogenic microorganisms, cyanobacteria is recognized as a proper platform for chemicals production due to their high growth rate. In this study, we aimed to develop an effective continuous process producing 1.3-propanediol (1.3-PDO) by genetically engineered cyanobacteria using an airlift bioreactor system. Firstly, to achieve PDO production in cyanobacteria, a synthetic metabolic pathway consisting of five genes were introduced into the host strain (PCC7942), which produces 1.3-PDO from DHAP through glycerol. Using the cyanobacteria (TA2984), successful 1.3 PDO production (0.7 mM) associated with cell growth was confirmed and the optimal culture conditions for 1,3-PDO production were pH8.0 and 220 (μE/m2/s). Based on this result, we examined the possibility of continuous process using TA2984. As a bioreactor for continuous process, an airlift fermenter with an internal draft tube was used because it achieves homogeneous distribution in a reactor with low hydrodynamic shear. Using the air-lift bioreactor system, The continuous 1,3-PDO production was performed for 40 days. However, its operation sometimes becomes unstable due to the increase of culture pH at high cell concentration. Therefore, we then introduced pH-stat system by CO2 supply. Using the pH-stat system, continuous 1,3-PDO production was achieved using air-lift bioreactor for 60 days and the highest 1,3-PDO productivity was increased to 0.227 (mM/day) at the dilution rate of 0.178 (day-1). Furthermore, it was found that glycerol addition to medium was effective to improve 1,3-PDO production. Based on these results, enhanced continuous production of 1,3-PDO using air-lift bioreactor with the productivity of 0.367(mM/day) was successfully performed for more than 60 days.
Methane has attracted attention in recent years not only as a clean alternative fuel for transportation vehicle but also as a chemical feedstock because it exists abundantly as the main component of natural gas, shale gas, and biogas. However, methane exists as a gaseous state at ordinary temperatures and pressures, making it expensive to store and transport from remote places where it is generated to places where it will be consumed. Therefore, efficient methodologies for converting methane to liquid fuels and/or valuable chemicals are required.
Methanotrophic bacteria have been considered to be a suitable host strain for the biological methane conversion because they are capable of utilizing methane as sole energy and carbon source. However, there are several obstacles in the use of methanotrophic bacteria for the biological methane conversion; for example, (1) slow growth rate, (2) slow mass transport of methane, (3) maintenance of intracellular redox balance, and (4) limitation of genetic manipulation tools. In this talk, we would like to introduce our achievements regarding (3) and (4). Methylococcus capsulatus (Bath) was used as a model methanotrophic bacterium. To maintenance of intracellular redox balance, the method for real-time monitoring of intracellular NADH:NAD+ ratio was established using a genetically encoded NADH sensor protein and electrochemical method (Bioresour. Techonol., 241, 2017, 1157-61). For a gene replacement, efficient counterselection method was proposed using a mutated pheS gene as a marker (Appl. Environ. Microbiol., 84, 2018, e01875-18). We anticipate that these our achievements will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.
Cellulosic biomass consisting of plant matter is the most abundant carbon-neutral renewable resource on the planet. Researchers from interdisciplinary fields have focused on biorefinery utilizing (ligno)cellulosic biomass for the production of bioenergy and biochemicals in recent years. To achieve the efficient utilization of cellulosic biomass as feedstock, the pretreatment step is one of critical issues to be solved (1, 2). To explore an alternative way to produce value-added biochemicals from biomass, we have focused on the potential of insect biorefinery in which a unique silkworm-baculovirus protein expression system is exploited in collaboration with Faculty of Agriculture in Kyushu University (3, 4). Herein, our efforts on combining the unique protein expression system and the enzymatic protein manipulation will be presented toward the sustainable production of functional proteins with mulberry leaves as a sustainable plant resource.
References
1) N. Kamiya, H. Takahashi et al., Biotechnol. Lett., 30, 1037-1040 (2008)
2) Uju, T. Oshima, M. Goto, N. Kamiya et al., Bioresour. Technol., 138, 87-94 (2013)
3) Patmawati, K. Minamihata, N. Kamiya et al., Biotechnol. J., 13, 1700624 (2018)
4) Patmawati, K. Minamihata, N. Kamiya et al., J. Biotechnol., 297, 28-31 (2019)
Development of rapid and powerful mutagenesis and high throughput adaptive evolution tools is of importance for creation of hyper cell factories by discovery of novel functional genes or biological dark matters and genome-phenotype association using integration of different approaches. ARTP (atmospheric and room temperature plasma) mutagenesis system developed by our group can directly cause complex genome mutation including chain break and bases mutation via a unique mechanism. Mechanistic study and various practical applications in cell breeding demonstrated that ARTP mutagenesis is apowerfultool. For developing an integrated platform capable of combing ARTP mutagenesis and high throughput adaptive evolution, we further developed a microdroplet-based microbial culture (MMC) system which can be operated automatically with high throughput culture on microchips, good repeatability, online detection of growth states, reprogrammable software, automatic addition of gradient chemical factors. The ARTP mutagenesis together with MMC system is an enabling platform for smart integrative biobreeding by further combining with genome editing technology, which can greatly contribute to green biomanufacturing.
This work is supported by National Key Scientific Instrument and Equipment Project of NSFC (2162780028), the Tsinghua University Initiative Scientific Research Program (20161080108) and the JST CREST Project of Japan.
