Marinagarivorans cellulosilyticus sp. nov., a cellulolytic bacterium isolated from the deep-sea off Noma-misaki, Japan.

Cells from strain GE09T, isolated from an artificially immersed nanofibrous cellulose plate in the deep sea, were Gram-stain-negative, motile, aerobic cells that could grow with cellulose as their only nutrient. Strain GE09T was placed among members of Cellvibrionaceae, in the Gammaproteobacteria, with Marinagarivorans algicola Z1T, a marine degrader of agar, as the closest relative (97.4 % similarity). The average nucleotide identity and digital DNA-DNA hybridization values between GE09T and M. algicola Z1T were 72.5 and 21.2 %, respectively. Strain GE09T degraded cellulose, xylan and pectin, but not starch, chitin and agar. The different carbohydrate-active enzymes encoded in the genomes of strain GE09T and M. algicola Z1T highlights their differences in terms of target energy sources and reflects their isolation environments. The major cellular fatty acids of strain GE09T were C18 : 1 ω7c, C16 : 0 and C16 : 1 ω7c. The polar lipid profile showed phosphatidylglycerol and phosphatidylethanolamine. The major respiratory quinone was Q-8. Based on these distinct taxonomic characteristics, strain GE09T represents a new species in the genus Marinagarivorans, for which we propose the name Marinagarivorans cellulosilyticus sp. nov. (type strain GE09T=DSM 113420T=JCM 35003T).

Microbial growth at hyperaccelerations up to 403,627 x g.

It is well known that prokaryotic life can withstand extremes of temperature, pH, pressure, and radiation. Little is known about the proliferation of prokaryotic life under conditions of hyperacceleration attributable to extreme gravity, however. We found that living organisms can be surprisingly proliferative during hyperacceleration. In tests reported here, a variety of microorganisms, including Gram-negative Escherichia coli, Paracoccus denitrificans, and Shewanella amazonensis; Gram-positive Lactobacillus delbrueckii; and eukaryotic Saccharomyces cerevisiae, were cultured while being subjected to hyperaccelerative conditions. We observed and quantified robust cellular growth in these cultures across a wide range of hyperacceleration values. Most notably, the organisms P. denitrificans and E. coli were able to proliferate even at 403,627 × g. Analysis shows that the small size of prokaryotic cells is essential for their proliferation under conditions of hyperacceleration. Our results indicate that microorganisms cannot only survive during hyperacceleration but can display such robust proliferative behavior that the habitability of extraterrestrial environments must not be limited by gravity.

Protocol for analyzing enzymatic hydrolysis of cellulose using surface pitting observation technology.

Assaying enzymatic degradation of water-insoluble substrates like cellulose is challenging because only the substrate surface is accessible to the enzymes resulting in low reaction rates. Here, we describe a protocol for surface pitting observation technology (SPOT), an ultra-sensitive quantitative assay for analyzing enzymatic hydrolysis of cellulose. We describe the use of a porous substrate to accelerate the hydrolysis rate of cellulose. We also detail the steps for combining inkjet patterning and optical profilometry to analyze volume loss upon hydrolysis. For complete details on the use and execution of this protocol, please refer to Tsudome et al. (2022).1.

An ultrasensitive nanofiber-based assay for enzymatic hydrolysis and deep-sea microbial degradation of cellulose.

Substrates for enzymatic reactions, such as cellulose and chitin, are often insoluble in water. The enzymatic degradation of these abundant organic polymers plays a dominant role in the global carbon cycle and has tremendous technological importance in the production of bio-based chemicals. In addition, biodegradation of plastics is gaining wide attention. However, despite the significance, assaying these degradation reactions remains technically challenging owing to the low reaction rate, because only the surface of the substrate is accessible to the enzymes. We developed a nanofiber-based assay for the enzymatic hydrolysis of cellulose. This assay facilitated the quantification of the enzymatic hydrolysis of <1 ng crystalline cellulose. Utilization of the assay for the functional screening of cellulolytic microorganisms revealed an unprecedented genetic diversity underlying the production of deep-sea cellulase. This study reiterates that interdisciplinary efforts, such as from nanotechnology to microbiology, are critical for solving sustainability challenges.

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

Versatile solidified nanofibrous cellulose-containing media for growth of extremophiles.

Solidified media that employ a porous matrix of nanofibrous cellulose are described. The physicochemical stability of the porous structure allows the development of solidified media that can support the growth of extremophiles, such as acidophilic Acidiphilium, alkaliphilic Bacillus, thermophilic Geobacillus and Thermus, alkalithermophilic Bacillus, and acidothermophilic Sulfolobus microbes. The cellulose-supported media have several advantages over agar- and gellan gum-derived media, including versatility and stability.

Suppression of the coffee-ring effect by sugar-assisted depinning of contact line.

Inkjet printing is of growing interest due to the attractive technologies for surface patterning. During the printing process, the solutes are transported to the droplet periphery and form a ring-like deposit, which disturbs the fabrication of high-resolution patterns. Thus, controlling the uniformity of particle coating is crucial in the advanced and extensive applications. Here, we find that sweet coffee drops above a threshold sugar concentration leave uniform rather than the ring-like pattern. The evaporative deposit changes from a ring-like pattern to a uniform pattern with an increase in sugar concentration. We moreover observe the particle movements near the contact line during the evaporation, suggesting that the sugar is precipitated from the droplet edge because of the highest evaporation and it causes the depinning of the contact line. By analyzing the following dynamics of the depinning contact line and flow fields and observing the internal structure of the deposit with a FIB-SEM system, we conclude that the depinned contact line recedes due to the solidification of sugar solution without any slip motion while suppressing the capillary flow and homogeneously fixing suspended particles, leading to the uniform coating. Our findings show that suppressing the coffee-ring effect by adding sugar is a cost-effective, easy and nontoxic strategy for improving the pattern resolution.

Preparation and characterisation of nanofibrous cellulose plate as a new solid support for microbial culture.

Porous plates made of nanofibrous crystalline cellulose were prepared, and used as a solid support for microbial cultures. Representative mesophilic microorganisms (, , and ) grew on the cellulose plate, just as well as they did on the conventional agar plate. optical microscopic examination revealed that the cellulose plate remained unchanged up to 280 °C at a constant pressure of 25 MPa. Due to the structural stability at high temperatures, a representative thermophile, , was cultured successfully on the cellulose plate at 80 °C. Mouse fibroblast cells did not show significant adhesion or extension on the cellulose plate.

An alkali-inducible flotillin-like protein from Bacillus halodurans C-125.

Flotillins are markers of lipid microdomains, and have emerged as a key concept in cellular biology. However, it remains unclear whether flotillin proteins exist in prokaryotic cells. The amino acid sequence of the BH3500 protein from Bacillus halodurans was 30% identical to that of flotillin-1. Motif analysis revealed that several specific residues (SPFH and flotillin domains, an AEA-repeat structure) and five potential phosphorylation sites are conserved in the BH3500 protein. In addition, the BH3500 protein was found to possess two transmembrane-spanning domains at the N-terminus, which is consistent with the common properties of flotillin-1. The BH3500 protein was detected in the Triton-insoluble, buoyant membrane fraction of B. halodurans by mass spectrometry and Western blotting. Interestingly, BH3500 was expressed strongly in alkaline conditions at both transcriptional and translational levels, which implies that it is one of the alkali-inducible proteins.