Hironaga Akita

The mitigation of anthropogenic climate change caused by fossil fuel combustion is a critical global challenge that necessitates a transition to renewable energy systems. Bioethanol represents a major renewable fuel, but first-generation production relies on edible feedstocks, which raises concerns regarding food security. Consequently, research is shifting toward second-generation bioethanol produced from abundant non-edible lignocellulosic biomass sources. This review comprehensively examines the potential of Candida krusei (synonyms: Pichia kudriavzevii, Issatchenkia orientalis) to serve as an alternative biocatalyst for second-generation bioethanol production. Compared with the first-generation bioethanol-producing yeast Saccharomyces cerevisiae, C. krusei exhibits superior physiological traits, such as thermo, acid, and inhibitor tolerances, enabling the utilization of several lignocellulosic feedstocks. This review summarizes the taxonomic and physiological characteristics of C. krusei, describes case studies on bioethanol production, and discusses strategies for reducing production costs. Furthermore, the technical and biosafety challenges associated with the industrial deployment of C. krusei are critically examined, including xylose metabolism limitations, scale-up constraints, and the management of its opportunistic pathogenic nature. A life cycle assessment perspective suggests that the unique physiological properties of C. krusei contribute to reducing greenhouse gas emissions and energy consumption throughout the entire production process, from pretreatment to downstream ethanol recovery.

The demand for d-amino acids is increasing as their unique physiological role continues to be elucidated. meso-Diaminopimelate dehydrogenase catalyzes the reversible NADP+-dependent oxidative deamination of meso-diaminopimelate to produce l-2-amino-6-oxopimelate. Moreover, a few enzymes show a broad range of reductive amination activity toward 2-oxo acids, synthesizing d-amino acids. Here, we report the identification and characterization of a novel NADP+-dependent meso-diaminopimelate dehydrogenase from Candidatus Syntrophocurvum alkaliphilum (CSaDAPDH). The purified enzyme exhibited oxidative deamination activity toward meso-diaminopimelate, as well as reductive amination activity toward several 2-oxo acids to produce the corresponding d-amino acids. The optimum pH and temperature for oxidative deamination of meso-diaminopimelate were 9.0 and 45°C, respectively. The optimum pH for reductive amination of pyruvate was 6.5, which was the lowest optimum pH among known enzymes, and thus, may lead to the development of new enzymatic methods for d-amino acid production. CSaDAPDH retained more than 60% of its activity after incubation for 30 min at 50 °C (pH 7.0) or at pHs ranging from 5.5 to 7.0 (50°C). Moreover, using known enzymes as comparisons, the coenzyme and substrate recognition mechanisms of CSaDAPDH were elucidated based on a multiple sequence alignment and the homology model.

Palladium, a non-toxic platinum-group metal, is widely used in catalysis, electronics, hydrogen storage, and chemical industries because of its excellent physical and chemical properties. However, given that the number of palladium-producing countries is limited, recycling is considered essential for ensuring a stable and sustainable global supply. Here, I describe a simple and efficient method for palladium recovery from electronic waste (e-waste) using Enterobacter oligotrophicus CCA6^T. To clarify biomineralization capacity, the role of electron donors in modulating biomineralization capacity was examined. Findings showed that formic acid was the most effective donor, enhancing the relative recovery rate to 44% compared to 23% without electron donors. Transmission electron microscopy analysis revealed palladium particles (1–10 nm) distributed across the cell wall, periplasmic space and cytoplasm, confirming active biomineralization rather than passive biosorption. Moreover, based on a comparison with the biomineralization mechanism of Escherichia coli, the biomineralization mechanism of E. oligotrophicus CCA6^T was estimated . Reaction parameters were then optimized by testing the effects of formic acid concentration, reaction temperature, and reaction pH. Under optimized conditions, the relative recovery rate exceeded 99% within 6 h using 40 mg/L palladium. When this method was applied to a metal dissolution solution prepared from e-waste , a recovery rate of 94% was achieved from trace concentrations (36 µg/L), and palladium loss from bacteria after the palladium recovery test was negligible (E. oligotrophicus CCA6^T could potentially be applied to the recovery of palladium from e-waste, particularly for trace-level concentrations where conventional methods are ineffective.

Interest in the production of bioethanol from inedible biomass is growing worldwide because of its sustainable supply and lack of competition with food supplies. Candida krusei (also known as Pichia kudriavzevii or Issatchenkia orientalis) is one of the most suitable thermotolerant yeasts used in the simultaneous saccharification and fermentation process for bioethanol production. In the production of bioethanol from lignocellulosic biomass as a feedstock, various environmental conditions occur, and the stress tolerance capacity of C. krusei, especially its low pH and tolerance to inhibitors, limits its practical application. In this study, to select a suitable second-generation bioethanol-producing strain, the tolerance capacity of five available C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) was characterized. Spot assay and growth experiment results showed that among the five C. krusei strains, C. krusei NBRC1664 showed superior tolerance capacity for low pH and inhibitors. Furthermore, this strain efficiently produced ethanol from glucose under low pH conditions with or without sulfate. A comparative analysis of the draft genome sequences suggested that Opy2, Sln1 and Cdc24 in the HOG pathway are conserved only in C. krusei NBRC1664, which may contribute to its superior tolerance to low pH levels. Moreover, amino acid sequence alignment showed that aldehyde dehydrogenase family proteins, which catalyze the degradation of cyclic aldehydes, are commonly conserved in C. krusei. In addition, the increased transcription levels in C. krusei NBRC1664 could play a role in its higher tolerance to inhibitors. These results suggest that C. krusei NBRC1664 is a more suitable strain for application in industrial processes for second-generation bioethanol production.

As isobutanol exhibits higher energy density and lower hygroscopicity than ethanol, it is considered a better candidate biofuel. The sustainable supply of inedible biomass and lack of competition with the food supply have stimulated significant worldwide interest in the production of isobutanol from this resource. Both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) have been applied to isobutanol production to effectively utilize inedible biomass as a feedstock. However, both processes have various challenges, including low isobutanol yield and high production costs. This review summarizes the potential of isobutanol as a biofuel, methods for conferring isobutanol productivity, recent experimental studies, and developments in both SHF and SSF with the isobutanol-producing strains. Challenges to increasing the isobutanol yield and various suggestions for improvements to enable commercial production are also discussed.

The thermotolerant yeast Pichia kudriavzevii (previously known as Issatchenkia orientalis), can produce ethanol from a variety of carbon sources and grows at around 45 °C. Thus, this yeast is considered a useful biocatalyst for producing ethanol from lignocellulose through simultaneous saccharification and fermentation (SSF). SSF has several advantages, such as a simplified manufacturing process, ease of operation and reduced energy input. Using P. kudriavzevii NBRC1279 and NBRC1664, we previously succeeded in producing ethanol through SSF; however, the extent to which inhibitors by-produced from lignocellulose hydrolysis affect the growth and ethanol productivity of the two strains remains to be investigated. In this study, to better understand the inhibitor tolerance capacity of the two strains, spot assay, growth experiment, real-time quantitative PCR (RT-qPCR) analysis and multiple sequence alignment analysis were carried out. When P. kudriavzevii NBRC1279 and NBRC1664, as well as Saccharomyces cerevisiae BY4742 as a control, were cultured on SCD plates containing 17% ethanol, 42 mM furfural, 56 mM 5-hydroxymethylfurfural (HMF) or 10 mM vanillin, only P. kudriavzevii NBRC1664 was able to grow under all conditions. Moreover, the inhibitor tolerance capacity of P. kudriavzevii NBRC1664 was greater than those of other strains using SCD medium containing the same concentrations of various inhibitors. When an RT-qPCR analysis of seven gene sequences from aldehyde dehydrogenase and the aldehyde dehydrogenase family protein (ADHF) was performed using P. kudriavzevii NBRC1664 cultivated in the presence of 56 mM HMF, ADHF1 and ADHF2 were up-regulated in the early logarithmic growth phase. Moreover, a multiple sequence alignment of the amino acid sequences of ADHF1, ADHF2 and the known ADH suggested that ADHF1 and ADHF2 may catalyze the reversible NAD⁺-dependent oxidation of HMF. Our data may be useful for future studies on the metabolic engineering of more useful strains for ethanol production from lignocellulose.

Each year, near 40 million tons of banana peels are discarded around the world. This plant biomass could potentially be utilized for energy production. Simultaneous saccharification and fermentation (SSF) is an effective method for producing biofuels from plant biomasses. Since SSF with enzymatic hydrolysis and fermentation are performed simultaneously in the same reactor, the production process is simpler than most existing methods. Here, we describe isobutanol production using SSF with hydrothermally treated banana peel samples and an Escherichia coli strain able to utilize glucose and xylose to produce isobutanol. To enhance the glucose and xylose concentrations, the reaction conditions for the enzymatic hydrolysis of plant biomass using two kinds of saccharification enzymes were optimized, including the enzyme unit ratio, reaction temperature and sample gram. When the optimized conditions for enzymatic hydrolysis were applied to SSF, the glucose and xylose produced from the hydrothermally treated samples were consumed, producing isobutanol. Moreover, the isobutanol concentration increased with an increasing initial culture pH, reaching 1.27 g/L at pH 6.5, which was consistent with the optimal initial culture pH for isobutanol production by this E. coli strain. Taken together, these results indicate that the established method is potentially useful for industrial isobutanol production.