As global climate change intensifies, forest fires are becoming more frequent and intense, Forest soil microbiomes have important ecosystem roles, such as sequestering soil carbon and nitrogen, processing soil nutrients, and forming critical interactions that support vegetation growth. This emphasizes the importance of studying the impact of fire on soil microbes. To achieve this goal, we conducted field surveys comparing two different pine species adapted to distinct fire regimes: a fire-tolerator (Pinus taiwanensis, subg. Pinus,台灣二葉松) and a fire-avoider (Pinus morrisonicola, subg. Strobus,台灣五葉松). We analyzed changes in soil properties and microbial communities before and after the fire, utilizing shotgun metagenomics technology to examine soil microbial communities. The results showed that the alpha diversity index significantly increased after the fire, especially for the Strobus site. At class level, there was a notable enrichment of Thermoleophilia, Ktedonobacteria and Gammaproteobacteria following the fire event. This suggested that these microorganisms may possess traits that enable them to adapt to new niches created by fire. Prior to the fire event, notably higher concentrations of NO3- and SO42- were observed at the Strobus site, compared to those at the Pinus site, while such differences were eliminated after the fire. These results suggested that the effects of fires on soil microbial communities are multifaceted, leading to short-term damage to certain microorganisms while simultaneously fostering the growth of others. Further exploration is ongoing to understand how soil microbiome changes thoughout the forest recovering process.

Symbiont switching often facilitates the rapid adaptation to new habitats and increases the survivorship in a changing environment. Wood-feeding stag beetles are known to associate with xylose-fermenting yeasts, which are transmitted vertically from mother insects to offspring via an exoskeletal symbiont-storage organ, called mycangium. In Taiwan, the stag beetle Neolucanus swinhoei distributes in wide range of altitudes, therefore, they may profit from symbiont switching to adapt different environmental temperatures. First, we isolated the yeast symbionts from mycangia of N. swinhoei from 37 sites in Taiwan, and those of some other stag beetles which may share the habitat with N. swinhoei. Phylogenetic analysis of IGS region showed that the yeast symbionts of N. swinhoei were placed in two distant monophyletic clades, namely, L and M. The clade L consisted exclusively of the yeasts of N. swinhoei in lowland populations, whereas the clade M included the yeasts of N. swinhoei in mountain populations and those of multiple Lucanus species, which also distribute in higher elevations in Taiwan. The maximum growth temperatures of L-type yeasts were significantly higher than those of M-type yeasts. Second, we tested the heat tolerance of the yeast symbionts in living adult females. Yeast CFU in the mycangium significantly decreased after the heat treatment (27℃, 7 days) compared to the control (20℃, 7 days) in the mountain populations, while no change was observed in the lowland populations. Third, long-term survivorship of the yeast symbionts was tested at the larval habitat. Liquid-cultured L- and M-type yeasts were mixed with wood sawdust in glass bottles and put in decaying wood of three field sites at different altitudes during the summer season. Consequently, CFU of both L- and M-type yeasts tended to decrease by decreasing altitude. Moreover, we confirmed that adult females of N. swinhoei re-acquire the mycangial symbionts from the pupal chamber at the moment of eclosion, suggesting that horizontal acquisition of symbionts may occur at this moment. Our study revealed that N. swinhoei is associating with two different yeast symbionts with different heat tolerance depending on altitude, and suggested that symbiont switching may occur under the changing environment. 

Due to overfishing, the population of anguillid eels has significantly declined worldwide. Compared to Anguilla anguilla and Anguilla japonica, information about the physiology of Anguilla bicolor is scarce. Studies showed that fish physiology is associated with host-associated microbiota. We aimed: 1) to study the gut and skin bacteria of eels at different life stages, and 2) to investigate the influence of eel sex hormones on the gut and skin bacteria. We collected freshwater Anguilla bicolor eels at different life stages (juvenile, immature, adult female, and adult male), measured their morphological traits, and quantified their hormone concentration. We show that the plasma estradiol concentration and the gonadal vtgr gene expression were significantly higher in females than in males. We collected a total of 126 samples, including intestinal fecal, skin mucus, fish farm water, and fish food samples (i.e., frozen bloodworms and pellets, extracted their genomic DNA, and performed full-length 16S rRNA amplicon PacBio sequencing. We show that older eels' (~3-year-old) gut bacterial diversity was significantly higher than younger eels (lower than 2-year-old), whereas the bacterial compositional structure of older eels was significantly different compared to younger eels. We also investigated which bacteria taxa contributed to the difference in bacterial compositional structure. By using the culturomics approach, we found 21 steroid-metabolizing bacterial species that could metabolize either estradiol or testosterone. All results show that steroid-metabolizing bacteria might play important roles in regulating sex hormones in tropical eels.

Most photosynthetic organisms, such as plants and algae, predominantly utilize the energy from the effective photosynthetic radiation (wavelength: 400-700 nm). However, certain specialized cyanobacteria have evolved a unique mechanism known as Far-Red Light Photoacclimation (FaRLiP), allowing them to harness lower-energy far-red light (wavelength: 700-800 nm) for photosynthesis. Purification of far-red light cyanobacteria from the environment contributes to a deeper understanding of this mechanism and its potential applications. Presently, research on far-red light cyanobacteria is limited, and the purification process is time-consuming and intricate, impeding further advancements in their study. In this study, it was discovered that antibiotics imipenem and novobiocin treatments can effectively increase the relative abundance of cyanobacteria. By testing environmental samples under different cultivation conditions, we aim to analyze which conditions result in an increased abundance of cyanobacteria. Initially, pretests on antibiotic concentrations and pH values were performed using the cyanobacterial model strain Synechocystis sp. PCC 6803 (Syn 6803) and the far-red light cyanobacterial strain Chlorogloeopsis fritschii PCC 9212 (Cf 9212). Subsequent experiments included antibiotic treatments (imipenem and novobiocin), pH10 treatment, and medium dilution 2-fold and 4-fold. The final analysis of 16S rDNA V3-V4 sequencing indicated that samples treated with imipenem and novobiocin antibiotics displayed the highest relative abundance of cyanobacteria, with minimal influence on the composition of far-red light cyanobacteria in the samples. These results provide diverse insights for future purification methods of far-red light cyanobacteria. Furthermore, our analysis covers all cyanobacteria and other bacteria, contributing to the purification of both general cyanobacteria and other bacteria. Future analyses in this study will focus on the inhibitory effects on other bacteria, with the aim of identifying conditions that not only increase cyanobacterial abundance but also inhibit the growth of other bacteria.

Photosynthetic organisms in natural habitats adapt to diverse light conditions. Species thriving in low-light environments, often sheltered beneath plants or rocks, utilize filtered light as their primary energy source. Filtered light is rich in underutilized far-red wavelengths. To overcome the constraints imposed by filtered light conditions, certain cyanobacteria and algae employ specialized photosynthetic pigments and antenna systems to effectively capture and utilize these far-red wavelengths. Through sampling the natural environment and cultivating it using far-red light (FRL, wavelength = 700-800 nm), I have isolated ten algal and six cyanobacterial strains that can grow in FRL from an urban park and Yuanyang Lake area in Taiwan. Among the isolated algae, four freshwater green algal genera have not been reported as capable of using FRL for photosynthesis. Compared to a model unicellular green alga, the ten isolated algal strains showed noticeable FRL absorbance features and increased FRL fluorescence emission at room temperature. However, unlike the six cyanobacterial strains that synthesize chlorophylls d and f to assist FRL harvesting, these algal strains only produce chlorophylls a or b. Therefore, additional red-shifted chlorophyll a molecules are likely associated with light-harvesting complexes in these algae. These green algae are not phylogenetically unique but closely related to other green algae in the same genera. However, it was not easy to discover that they can grow in FRL without growth validation, which might be why they were not previously reported capable of using FRL. Overall, our results suggest that FRL-using algae may be widespread, and their diversity may be underestimated in the environment.

Internal ribosome entry sites (IRES) permit the recruitment of the ribosomal preinitiation complex to the mRNA initiation codon without the involvement of the 5’end. It has been widely reported in eukaryotic cells and viruses, but there are few studies to address the distinct types of IRES elements in the genome of bacteriophages. The intergenic region (IG) (NcoI 6043-PstI 4847) of bacteriophage Cf contains motifs with predicted secondary structures, such as positive and negative replication origins, and the phage integration site. To assess whether the Cf-IG region carries IRES activity, PCR amplification of three segments within the Cf-IG region, namely Cf-IG-seq1, Cf-IG-seq2, and Cf-IG-seq3, had previously been performed in luciferase reporter plasmids to evaluate their effect on luminescence expression. The results indicate that the Cf-IG-seq3 element possesses the highest potential for functioning as an IRES. In this study, our aim is to conduct sequence deletion analysis on the seq3 sequence in order to identify the minimal segment with IRES activity. We also aimed to construct a circular RNA vector containing the seq3 sequence and a fluorescent protein, generating circular RNA through the 'backsplices' mechanism without a 5' cap. Based on the property of IRES-mediated cap-independent translation, we examined the capability of seq3 to recruit ribosomes for translation and expression of the GFP protein. Experimental results demonstrated that the shortest IRES-active segment of seq3 is 55 nucleotides (nucleotides 113-168). Furthermore, fluorescence microscopy observations revealed successful GFP protein expression from the forward-oriented seq3 sequence, whereas the reverse-oriented sequence exhibited weaker signals. 

In recent years, the massive use of plastics and their improper disposal have led to significant pollution in the oceans. Currently, over 1.6 million square kilometers of plastic trash, equivalent to 230 million soccer fields, floats in the sea. This waste provides a platform for numerous organisms and microorganisms to colonize, a plastic associated ecosystem now known as the “plastisphere”. In addition, studies have shown that plastispheres have the ability to attract marine organisms and lead to uptake of marine organisms, which may be related to the production of syngeneic microorganisms from chemical metabolites produced on the surface of the plastic. Environmental DNA (eDNA) is the genetic material left behind by organisms in the environment and has been recognized as a method of monitoring the presence of organisms in the environment. Therefore, in this study, we prepared plastic bottles and incubated it in situ at a semi-open coastal habitat for various time points. We collected water from the habitat, both with and without plastic bottles, for eDNA detection and analyzed the microorganisms colonizing the plastic surfaces using Oxford Nanopore technology. Additionally, we examined the microbial metabolites produced by these microorganisms using liquid chromatography-mass spectrometry to explore the key microbial metabolites that attract copepods. Results showed that the microorganisms on the surface of the plastic were attracted to copepods and that the microbial composition varied significantly at different time points. Analysis of the eDNA results revealed that certain species were attracted to the plastic surface at specific time points. However, the correlation between these findings and the profiling of chemical metabolites requires further exploration.

果膠酶一般多用於食品加工，目前研究指出分解植物細胞壁的同時可刺激植物免疫。因而本研究從丁酸梭菌中選殖二種不同的果膠酶基因，分別是多聚半乳糖醛酸酶(polygalacturonase ;PG)、果膠裂解酶(pectin lyase ;PL)。於此研究中將二種果膠基因轉型至pET21a載體中，並於E.coli BL21 (DE3)中大量表達，再將所得重組蛋白、酵素進行活性測試及表現量測試。從丁酸梭菌的基因裡設計出二種果膠酶引子對，以PCR擴增出預期的PG與PL之DNA片段分別為1523 bp及961 bp。將PCR產物純化並轉入T&A vector，使用限制酶Hind III切割，確認位置大小後，將其送定序，並將序列至NCBI作比對。確認無誤後，再將PG和PL基因分別轉殖入pET21a載體中，並使用對應設計含BamH I、Xho I限制酶之引子對進行PCR擴增、限制酶切割等確認。PG與PL片段轉入pET21a載體後分別PG為5443bp及1523bp、PL為5443bp及961bp。經SDS-PAGE分析結果，可分別得到與預測之蛋白質PG分子量為60 kDa、PL分子量為34 kDa相同之結果。現已成功將果膠酶表現出，未來可量化生產用於生物性非致病性微生物酵素提升植物免疫取代化學農藥。

Some special cyanobacteria can utilize energy in the far-red light region (FRL, λ=700~800nm) by performing far-red light photoacclimation (FaRLiP), which produce far-red light absorbing photosystem complexes that contain special chlorophylls (Chls) such as Chl d and Chl f. FRL-type Photosystem II (FRL-PSII) in FaRLiP strains contains subunits differ from white light (WL)-type. The conserved residue Tyr191 in the FRL-type subunit psbD3 binds to the reaction center of FRL-PSII, which potentially alters the electronic structure and helps accommodate FRL-specific pigment. However, there is still lacking in vivo evidences to support such hypothesis, and no point-mutated strain has been successfully created in FaRLiP cyanobacteria. Here we applied a CRISPR-Cpf1 system to perform point-mutation in psbD3 in a FaRLiP strain Synechococcus sp. PCC 7335 (Syn7335) and successfully generated Y191W mutation in PsbD3 subunit. Furthermore, we showed that the Tyr191 is an essential residue for photosynthesis under FRL. A WL-type residue were introduced into PsbD3 through conjugative transformation and CRISPR-Cpf1 gene editing system. The Y191W mutant showed diminished growth and altered pigment composition under FRL compared to WT. The absorption spectrum of mutant grown under FRL showed enhanced WL-type phycobilisome absorption, while the RT fluorescence spectrum showed enhanced Chl a signal, indicating that extensive expression of WL-type PSII was required for the mutant to survive under FRL. Furthermore, an emerging emission at 720nm in 77K fluorescence spectrum was observed, which has been reported as the emission of bulk allophycocyanin. The results demonstrated the utility of CRISPR-Cpf1 system in gene editing of Syn7335, and showed Tyr191 to be an essential residue for the function of FRL-PSII in Syn7335. Our work expands the gene editing tool of a FaRLiP strain and helps understanding the mechanism of FRL-using photosystem. Future work will focus on measuring other phenotype of the Y191W mutant such as oxygen evolution, as well as study the conserved residues in other FRL-PSII subunits using the CRISPR system.

Partial nitritation/anammox is a sustainable biotechnology to treat ammonium wastewater. However, because of imprecise bubble aeration to supply oxygen for partial nitritation, its application to treat low-strength ammonium wastewater is challenging. This study established a membrane oxygenation biofilm reactor to perform the partial nitritation/anammox process for treating low-strength ammonium wastewater. The breathable membrane supplies oxygen from the dry side with low energy consumption but high oxygen transfer efficiency to the biofilm attached to the wet side of the membrane. After optimizing oxygenation parameters, including duration, frequency, and flowrate of air supply, the reactor achieved an efficient nitrogen removals of >70% when treating synthetic wastewater with input ammonium-nitrogen of 80 mg/L. The aerobic ammonium oxidation rate of 3.82 mgN/gSS/hr was substantially higher than the aerobic nitrite oxidation rate of 0.1 mgN/gSS/hr, suggesting a successful partial nitritation of ammonium through precise membrane oxygenation. Redundancy analysis revealed that the anammox rate was positively correlated with aeration frequency but negatively correlated with aeration duration. The reactor was then tested with industrial wastewater at a loading of 80 mgNH4+-N/L/day, achieving high COD and nitrogen removals with 50% and 75% efficiencies, on average, respectively. Sequencing of 16S rRNA gene amplicons showed that relative abundances of nitrifying and anammox bacteria in the membrane-attached biofilms were 1.2 and 3.4 times higher than suspended sludge and 1.3 and 3.2 times higher than wall-attached biofilms, respectively. These results demonstrated that the membrane oxygenation biofilm reactor is a promising model for implementing a partial nitritation/anammox process to treat low-strength ammonium wastewater.