工業活動和現代化生活模式，加劇影響陸地與土壤環境系統中溫室氣體（Greenhouse gases, GHGs）排放，如二氧化碳（Carbon dioxide, CO2）、甲烷（Menthane, CH4）以及氧化亞氮（Nitrous oxide, N2O）的上升，增強了溫室效應，造成生態環境改變。生物炭 (biochar)是在低、無氧環境下熱裂解之含碳固體物質，其高比表面積、孔隙、良好吸附能力 (豐富官能基) 以及營養物質，可以有效改善土壤性質、微生物的活性、降低肥料及土壤養分的損失與減輕土壤污染，為了有效降低甲烷溫室氣體排放，在農業、養殖漁業、掩埋廠以及濕地等土壤環境中添加生物炭，由於高孔隙率的特性，有效增加土壤通透性，以利於土壤中甲烷氧化菌生長，提升其氧化甲烷之能力，並達到降低甲烷溫室氣體的排放。在不同生物炭添加比下，透過Gompertz動力學模型擬和CO2排放與CH4排放，並進一步對溫室氣體最大產生速度進行比較與探討。本研究 (1) 探討生物炭與土壤含水 (飽和與未飽和) 在比例0%、5%、10%、25%、50%以及100 % 情況下，CH4氣體排放之關係；(2) 探討生物炭與土壤含水 (飽和與未飽和) 在比例0%、5%、10%、25%、50%以及100 % 情況下，CO2之關係；(3) 透過Gompertz動力學模型探討在最佳生物炭10% 比例下，相較於未添加生物炭，降低17.24% (飽和土壤)、16.26 (未飽和土壤) 之CH4溫室氣體之最大產生速度；(4) 透過Gompertz動力學模型探討在最佳生物炭10%比例下，相較於未添加生物炭，降低26.99% (飽和土壤)、13.74% (未飽和土壤) CO2溫室氣體之最大產生速度。

Tap water, a source we regularly utilize in our daily lives, poses a significant health concern for society on its quality, and the soil run off is the main source of tap water. However, there are plenty of pathogens and hazardous substances in soil run off; to deal with them, various disinfection methods have been developed, and chlorination stands as a prevalent method in current tap water treatment processes due to its effectiveness. As global average temperatures rise, surface soil temperature increases accordingly. This temperature increase may alter the microbes in soil surface, changing the composition of organic matters and potentially escalate the toxicity of soil run off after chlorination treatment. This study using cultivated soil to be experimental samples subjected to different temperature conditions to explore the effects of temperature variations on the organic composition and concentration in surface soil, as well as the toxicity of soil run off after chlorination treatment, using the nematode, Caenorhabditis elegans (C. elegans), as a model organism for toxicity testing. After chlorination by uniform formation condition (UFC) method, it was observed that the chlorine demand curves for soil run off at 25°C and 35°C were generally similar, while the chlorine demand was lower for soil run off at 45°C. Regarding the reproductive toxicity in C. elegans, it is designed to a total of six experimental groups involving soil run off at 25°C, 35°C, and 45°C, both untreated and treated in chlorination with UFC method. The objective was to observe whether the reproductive capacity of C. elegans was affected after exposure. Each group consisted of at least 10 samples and underwent three biological replicates. Results indicated no reproductive toxicity at 25°C, regardless of chlorination. However, at 35°C and 45°C, untreated samples showed no reproductive toxicity, whereas chlorination resulted in reproductive toxicity. As for the locomotive toxicity in C. elegans, six experimental groups were established using soil run off at 25°C, 35°C, and 45°C, untreated and treated in chlorination with UFC method. The aim was to observe whether the locomotive ability of C. elegans was impacted after exposure. Each group had at least 10 samples and underwent three biological replicates. The findings indicated no locomotion toxicity across all conditions. However, despite not reaching statistical significance, a slight decline in locomotive behavior was observed in chlorinated samples at 35°C and 45°C. In conclusion, an increase in surface soil temperature might lead to the reproductive toxicity of soil run off after chlorination rises. Ammonia is a well-known aquatic nitrogenous pollutant that threatens fish health, particularly in aquaculture. The fish skin with mucus and residing microbiome serve as the primary external interface between the fish and host.

Ammonia has long been considered a toxic reagent to fish in the aquatic ecosystem. Our previous study has confirmed the protective role of fish skin mucus against ammonia impact, potentially through ammonia utilization by the mucus commensal bacteria. However, the potential bacterial species and their mechanisms behind ammonia utilization remain unexplored. The present study aims to investigate ammonia-utilizing bacteria (AUB) in the fish skin mucus and evaluate how these AUB utilize ammonia. The potential AUB were isolated by the fish skin mucus culturing with Ammonia-Enriched Medium (AEM). Species of the isolates were identified using 16S rRNA gene sequencing. AUB property was characterized by the ammonia utilization assay and nitrification activity was assessed by measuring the nitrate concentration within. We found a total of four isolates within the AEM-based culture. The four isolates were identified as Stenotrophomonas sp., Delftia sp., Agrobacterium sp., and Acinetobacter sp, respectively. Further characterization found that only Acinetobacter sp. can utilize ammonia and exhibit a utilization rate of up to 85% within a 5-day incubation. Furthermore, nitrate was produced and increased, corresponding to ammonia utilization of Acinetobacter sp. In conclusion, these data suggest that Acinetobacter sp. is one of the potential AUB in fish skin mucus. Moreover, this bacterium can utilize ammonia through nitrification. Future work on ammonia assimilation will be examined to determine the ammonia utilization process in the fish skin mucus. This study gives insight into the ammonia utilization in the fish skin mucus, potentially leading to the development of an application for aquatic animals and water quality control in the future. 

Swietenia macrophylla King (大葉桃花心木), commonly known as mahogany, is a high-quality wood with high economic value. It is resistant to high temperatures and droughts, making it very suitable for cultivation in Southern Taiwan. Mahogany is a large deciduous tree that annually sheds many leaves around March and April. When fallen leaves are decomposed through the action of microorganisms, mineral nutrients are released, contributing to the replenishment of moisture for the tree’s growth. In this study, we aim to understand the interaction between mahogany leaf litter and soil microbes in Xinhua Forest Area, using these changes as indicators for microbial activity. Therefore, we sampled surface soil at three time points: before leaf fall (February), the early stage of litter decomposition (July), and the late stage of litter decomposition (November). We analyzed the changes in soil properties and assessed the microbial community using V3-V4 region of 16S rRNA gene at the three time points. The results indicated that the soil pH remained consistently around 6, with no significant differences observed between seasons. However, the electric conductivity (EC) values showed a significant decrease in July. In addition, we measured soil ion concentrations and observed similar trends to the EC results. The concentration of cations (Na+, K+, Mg+, Ca+) and anions (NO3-, SO42-) exhibited significant decrease in July. In the microbial community changes between February and July, Alphaproteobacteria showed a decreasing trend, while Thermoleophilia showed an increasing trend. These results show that the impact of mahogany leaf litter on soil properties and microbial communities is gradual. Comparing results across various forest types allow us to better understand the contribution of mahogany leaf litter to the soil, providing insights for afforestation directions.

Biodegradation has long been a conventional strategy for remediating sites contaminated with chlorinated solvents or aromatic hydrocarbons. However, traditional remediation methods, whether anaerobic or aerobic, often fall short when dealing with co-occurring pollutants. In our study, utilizing trichloroethylene and toluene as model compounds, we explored an innovative remediation approach by manipulating anaerobic consortia with intermittent micro-oxygenation to achieve codegradation. Our results demonstrated that while oxygen inhibited the anaerobic dechlorination of trichloroethylene, the dechlorination rates remained surprisingly comparable to those observed at dissolved oxygen levels of 0.2 mg/L. Periodic micro-oxygenation induced fluctuations in reactor redox conditions within the anoxic and strictly anaerobic range, facilitating rapid degradation of the dual pollutants. Notably, trichloroethylene degradation accounted for only 27.5% of the non-inhibited dechlorination. To gain deeper insights into the microbial dynamics responsible for this degradation, we employed metabarcoding of nearly full-length 16S rRNA gene amplicons. Our analysis revealed the predominance of Dehalogenimonas (16.0% ± 3.5%) over Dehalococcoides (0.3% ± 0.2%), with tenfold higher transcriptomic activity in Dehalogenimonas. Shotgun metagenomics further unveiled numerous genes related to reductive dehalogenases and oxidative stress resistance in both Dehalogenimonas and Dehalococcoides. Additionally, we observed the enrichment of diverse facultative populations harboring functional genes associated with trichloroethylene co-metabolism as well as aerobic and anaerobic toluene degradation. These findings suggest that the codegradation of trichloroethylene and toluene involves a multitude of biodegradation mechanisms. Notably, the prevalence of genes encoding high-affinity terminal oxidases in these functional populations elucidates their survival strategy in the micro-oxygenation environment. In conclusion, our study demonstrates the efficacy of intermittent micro-oxygenation in facilitating the degradation of trichloroethylene and toluene. These findings hold promise for the bioremediation of sites contaminated with similar organic pollutants, offering a sustainable and efficient approach to environmental cleanup.

Although sex hormones, namely androgen and estrogen, were thought to produced exclusively by vertebrates and may appear after the Cambrian explosion, sterols can be biosynthesized under microaerobic conditions and are proposed to appear on earth 2.3 billion years ago. A variety of aerobic and anaerobic prokaryotes can degrade the side-chain of sterols through β-oxidation, remaining androgens as end products. One can thus envisage that androgens could appear soon after sterols were produced. Here, we report that strain TUW77, an acetogen isolated from the gut of great blue-spotted mudskipper (Boleophthalmus pectinirostris), can transform testosterone into estrone and 17β-estradiol through the Wood-Ljundahl pathway; the resulting androgenic C-19 methyl group is used as carbon and electron donors for bacterial growth. Physiological exams indicated that the strain TUW77 exclusively grows with testosterone, with estrogens as extracellular end products. The strain TUW77 genome contains two copies of a polycistronic gene cluster, aetABC (Anaerobic EsTrogenesis), which respectively encode the MT1, CoP, and MT2 components of a cobalamin-dependent methyltransferase and were highly expressed under testosterone-fed conditions and. Consistently, we observed the apparent production of AetABC in the testosterone-grown bacterial cells. Surprisingly, the primary structures of the MT1 (AetA) and CoP (AetB) components are most similar to the characterized EmtAB from the denitrifying Denitratisoma sp. strain DHT3 capable of anaerobically transforming estrogen into androgen (the reverse reaction). The identification of the cobalamin-dependent estrogenesis in acetogens represents an unprecedented metabolic link between steroid hormones biosynthesis and ancient C1 metabolism through the Wood-Ljundahl pathway.

Agrobacterium-mediated transformation by floral inoculation (AMT-FI) is a critical tool for studying gene functions in the model plant Arabidopsis thaliana. Although the system is well established, detailed mechanisms of several key steps are still unknown, which limits knowledge-based improvements. Moreover, nearly all disarmed strains were derived from the wild-type strain C58 and share the same chromosomal background. To harness the biological diversity of agrobacteria for basic research and potential applications, we developed a high-throughput phenotyping platform to evaluate the AMT-FI efficiency of diverse strains. The method utilizes the RUBY reporter system, which converts tyrosine in plant cells to betalain upon successful transformation, producing transformants with red colors that can be quantified based on our customized image analysis pipeline. We utilized this platform to evaluate 64 wild-type strains and 4 disarmed strains that belong to 3 genera and 10 species. Together, these strains harbor 11 types of oncogenic plasmids and cover most of the known genetic diversity of agrobacteria. Based on > 10 biological replicates per strain, 5 strains belonging to 4 different species were found to have statistically higher efficiencies than the reference strain C58. For validation, we tested those 5 high-efficiency strains based on the standard hygromycin resistance assay of seedlings to verify T-DNA integration, and confirmed that 4 of those strains performed better than C58. In summary, this work developed a high-throughput platform for assessing AMT-FI efficiency, which can be used for expanded sampling. Moreover, the high-efficiency strains identified can be used for future research on AMT-FI.

Coastal regions facing trichloroethene (TCE) contamination encounter a significant challenge in the realm of bioremediation. This challenge arises from the sensitivity of key organohalide-respiring bacteria, such as Dehalococcoides and Dehalogenimonas to salinity fluctuations. In our laboratory, we have successfully enriched anaerobic microbial consortia capable of completely dechlorinating TCE to ethene under saline conditions (7.62 grams per liter). To delve into the efficiency of TCE dechlorination under salinity variations, the abundance of crucial organohalide-respiring bacteria, and the dynamics of microbial communities under salinity variations, we established three batch reactors inoculated with the dechlorinating consortium. These reactors represent conditions of reduced salinity (SD), increased salinity (SI), and maintaining constant salinity followed by a sudden increase (SM). We assessed TCE dechlorination efficiency through GC-FID analysis, revealing that TCE can still undergo reductive dechlorination at salinity levels of 20-30g/L. Subsequently, we employed 16S rRNA amplicon long-read sequencing and RT-qPCR to unveil the dynamic changes in microbial community structure and the activity of key microorganisms and genes. Detailed results of this study will be presented and extensively discussed on MiTalk 8.

Bacteria, commonly known as unicellular organisms, can often exhibit surprising collective behaviors. Our research focuses on the bacteria Bacillus subtilis, which can develop from single cells into differentiated multicellular communities called biofilms. Previous studies have shown that biofilms can spatially pattern their cell differentiation into concentric rings. Furthermore, biofilms pattern these concentric rings by triggering a cell-autonomous oscillation of the nitrogen stress response. However, oscillations in individual cells are generally noisy and would struggle to form a coherent collective pattern unless they are synchronized. Is there a mechanism that synchronizes cell-autonomous oscillations? B. subtilis has been shown to generate synchronized electrochemical signals across the biofilm by changing the membrane potential in bacterial cells. Moreover, membrane potential could affect the uptake of nitrogen sources and possibly nitrogen stress response. Here we show that in a biofilm, signals of membrane potential correlate spatially and temporally with the concentric rings of nitrogen stress response. We develop new microfluidic chip designs for time-lapse microscopy to study the synchronization mechanism at the single-cell level. Our study aims to unravel the intricate interplay between developmental patterning and electrochemical signaling within bacterial biofilms through this interdisciplinary approach.  

Phycobilisomes (PBS) are antenna megacomplexes that transfer energy to photosystems II and I in thylakoids. PBS likely evolved from a basic, inefficient form into the predominant hemidiscoidal shape with radiating peripheral rods. However, it has been challenging to test this hypothesis because ancestral species are generally inaccessible. Here we use spectroscopy and cryo-electron microscopy to reveal a structure of a “paddle-shaped” PBS from a thylakoid-free cyanobacterium that likely retains ancestral traits. This PBS lacks rods and specialized ApcD and ApcF subunits, indicating relict characteristics. Other features include linkers connecting two chains of five phycocyanin hexamers (CpcN) and two core subdomains (ApcH), resulting in a paddle-shaped configuration. Energy transfer calculations demonstrate that chains are less efficient than rods. These features may nevertheless have increased light absorption by elongating PBS before multilayered thylakoids with hemidiscoidal PBS evolved. Our results provide insights into the evolution and diversification of light-harvesting strategies before the origin of thylakoids. 

Most cyanobacteria contain chlorophyll a as their major pigment in their photosystems, which absorbs red light (absorption peak: 665 nm) for photosynthesis. A unique marine cyanobacterium, Acaryochloris marina (A. marina), contains up to 95% or more chlorophyll d (absorption peak: 696 nm) in its photosystems, enabling the utilization of white light (WL) and far-red light (FRL) for photosynthesis. Most studies on A. marina have predominantly focused on the structural and mechanistic aspects of the photosystems. However, there are few studies delving into the responses of A. marina under various light conditions. Here we show that A. marina has advantages when growing under FRL conditions than WL conditions. We found that the doubling time of A. marina under FRL is shorter than that of the WL condition. The data of chlorophyll fluorescence experiments show that A. marina produces more nonphoto-chemical quenching (NPQ) under FRL, which is the mechanism of preventing photodamage. However, the maximum quantum efficiency of photosystem II (Fv/Fm) remains consistent between WL and FRL conditions, while the overall chlorophyll fluorescence of A. marina under FRL is higher than that of the WL condition. For the absorption and fluorescence spectra, there are only nuances between WL and FRL conditions. Pigment analysis reveals no significant differences in the chlorophyll d content. These results demonstrate that A. marina grows faster under FRL than WL, but the key factors are neither the chlorophyll d content nor the maximum quantum efficiency of PSII. This study sets the stage for further exploration into the underlying mechanisms governing the growth advantages of A. marina under FRL. Notably, the current data falls short of explaining these observations, prompting the need for additional investigations, including assessing the oxygen evolution rate and employing RNA sequencing to unravel the intricate gene interactions that shape the responses of A. marina under varying light conditions.

Cyanobacteria typically absorb Photosynthetically Active Radiation (PAR) within 400-700 nm. Occasionally, some cyanobacteria can harness far-red light (wavelength = 700-800 nm) beyond PAR limits. Despite the potential applications of far-red cyanobacteria, their natural habitats and diversity remain unclear. These organisms are predominantly isolated from hot springs, indicating the rich diversity within extreme environments. Notably, no reports exist of far-red cyanobacteria in the abundant hot spring environments in Taiwan. Our study of 37 samples from six Taiwanese hot springs fills this gap by exploring the distribution and analyzing microbial communities through 16S rRNA gene sequencing of the V3-V4 region. The results demonstrate that far-red cyanobacteria make up 1-30% of the total cyanobacterial population, with the identification of eight distinct OTUs, primarily belonging to the Leptolyngbya and Calothrix genera. The composition of far-red cyanobacteria and bacteria varied among sampling regions. The relative abundance of far-red cyanobacteria in hot spring samples significantly increases with pH. It slightly decreases with temperature, suggesting that the temperature range of 34 to 63°C may not substantially determine their abundance. The 16S rRNA gene sequencing results reveal a higher relative abundance of far-red cyanobacteria in Qikeng and CingShui hot springs. Additionally, HPLC pigment analysis detected chlorophyll f presence in these field samples, suggesting that far-red cyanobacteria utilize far-red light for photosynthesis in hot springs. Cultivation under far-red light revealed five far-red cyanobacteria, including aggregate and filamentous forms, and additional Chroococcidiopsis sp. not detected by 16S rRNA gene sequencing, suggesting greater diversity than recognized. These findings expand our understanding of far-red cyanobacterial diversity and provide a basis for studying thermophilic microbial communities in the neutral to alkaline hot springs of Taiwan.

Agrobacterium tumefaciens is a tool for genetic engineering based on its ability of transferring T-DNA on its tumor-inducing plasmid (pTi) to the host genome. Although A. tumefaciens strains are diverse in genome and virulence, the disarmed strains used in Agrobacterium-mediated transformation are mostly derived from strain C58. In our characterization of 30 wild-type A. tumefaciens strains, the strain with best transformation ability, 1D1108, is 3.8- fold stronger than C58. However, this phenotype does not correlate to gene content similarity. To characterize the genes that contribute high virulence, we conducted RNA sequencing to 1D1108 under acid or acetosyringone (AS) induction in vitro, and to 1D1108 isolated from infiltrated Nicotiana benthamiana. ~1,300 differential expression genes (DEGs) genes were identified in planta, which is around 10-fold larger than the DEGs between in vitro conditions. Beside the known acid or AS regulated genes, the role of other plant regulated genes in infection are mostly unclear. Further transcriptome analysis revealed the difference between the in vitro and in planta condition in gene expression pattern. In addition, differences were found when comparing 1D1108 dataset to published C58 transcriptome dataset. These results suggest that 1D1108 might have some different strategies during infection, and in planta transcriptome could provide us a comprehensive information of gene regulation during Agrobacterium infection. More in planta transcriptome dataset will be collected for comparison and further investigations of putative candidates are required to confirm their roles and mechanisms. The knowledge may be used for future synthetic biology work to improve Agrobacterium-mediated transformation.

In synthetic biology, light-inducible promoter systems offer precise control of heterologous gene expression with spatial and temporal patterns. Instead of toxic and costly inducers, light-inducible promoters are eco-friendly and cost-effective on an industrial scale. In our previous study, a far-red light (FRL) inducible promoter system RfpA/B/C-PchlFJSC1 was identified in cyanobacterium Chlorogloeopsis fritschii PCC 9212 (Cf9212). FRL-inducible promoters potentially control gene expression in deep layers of cells. However, the lack of characterization of the molecular mechanism prevents the RfpA/B/C-PchlFJSC1 system from being transferred to other organisms. This study aims to elucidate the FRL-inducible promoter-DNA interaction, the RfpA/B/C protein activation mechanisms, and the potential industrial application of the RfpA/B/C-PchlFJSC1 system in Cf9212. The RfpB transcription factors will be overexpressed in Cf9212, and the protein phosphorylation patterns will be validated in white light and FRL with Phos-tag gel. Meanwhile, the phosphorylation of RfpB and the potential interaction between RfpB and other proteins will be detected using mass spectrometry. In addition, the promoter-transcription factor interaction will be revealed by performing an electrophoretic mobility shift assay. At the same time, the FRL-regulated promoters in Cf9212 will be identified by chromatin immunoprecipitation sequencing. Furthermore, a multi-layer continuously-reacting photobioreactor will be constructed using the Cf9212 strain with FRL-inducible promoters. So far, the specific growth rate of Cf9212 in high cell density is characterized and the RfpB-overexpressing plasmid is constructed. These approaches enable the broad application of RfpA/B/C-PchlFJSC1 in diverse organisms with optimized promoter sequences, well-characterized protein systems, and a land-saving cost-effective bioreactor.

Lignocellulose includes cellulose, hemicellulose, lignin and traces of water-soluble pectins, proteins and aliphatic acids. Lignin consists of the non-polysaccharide portion of lignocellulosic biomass, is polymerized from phenylpropane and provides mechanical strength in plants. Some microorganisms have the ability to break down ether carbon-oxygen bonds and carbon-carbon bonds, which may help to degrade lignin in a more efficient way. In this study attempts were made to explore lignocellulolytic enzymes produced by soil bacteria. Three soil extract-based media containing plant constituents were used for the isolation of acidotolerant bacteria from tea plantations. Their 16S rDNA sequences and capabilities to produce several enzymes involved in lignocellulose degradation were also determined. During an 8-month cultivation period, a total of 100 isolates which belonged to bacterial families namely Burkholderiaceae, Chitinophagaceae, Kaistiaceae, Nocardioidaceae, Sphingomonadaceae and Streptomycetaceae were obtained. More than 40% of the isolates showing sequence similarity less than 98% compared with their most closely-related type strains as revealed in EzBioCloud database. These isolates were found to produce cellulase, xylanase, laccase, lignin peroxidase and manganese peroxidase. In addition to bacterial phyla Actinomycetota, Bacillota and Pseudomonadota (alpha- and gamma-Proteobacteria) which have been reported as lignocellulose-degrading bacteria, the lignocellulolytic enzymes activities were also demonstrated in beta-Proteobacteria (Pseudomonadota) and Chitinophagia (Bacteroidota) in this study. The soil extract-based media containing plant constituents were successfully used in the isolation of many novel bacterial species from natural environments, and their potential to produce novel enzymes can be further studied in a near future.