A hybrid photocatalytic system enables direct glucose utilization for methanogenesis.

Integration of methanogenic archaea with photocatalysts presents a sustainable solution for solar-driven methanogenesis. However, maximizing CH4 conversion efficiency remains challenging due to the intrinsic energy conservation and strictly restricted substrates of methanogenic archaea. Here, we report a solar-driven biotic-abiotic hybrid (biohybrid) system by incorporating cadmium sulfide (CdS) nanoparticles with a rationally designed methanogenic archaeon Methanosarcina acetivorans C2A, in which the glucose synergist protein and glucose kinase, an energy-efficient route for glucose transport and phosphorylation from Zymomonas mobilis, were implemented to facilitate nonnative substrate glucose for methanogenesis. We demonstrate that the photo-excited electrons facilitate membrane-bound electron transport chain, thereby augmenting the Na+ and H+ ion gradients across membrane to enhance adenosine triphosphate (ATP) synthesis. Additionally, this biohybrid system promotes the metabolism of pyruvate to acetyl coenzyme A (AcCoA) and inhibits the flow of AcCoA to the tricarboxylic acid (TCA) cycle, resulting in a 1.26-fold augmentation in CH4 production from glucose-derived carbon. Our results provide a unique strategy for enhancing methanogenesis through rational biohybrid design and reprogramming, which gives a promising avenue for sustainably manufacturing value-added chemicals.

Respiration-driven methanotrophic growth of diverse marine methanogens.

Anaerobic marine environments are the third largest producer of the greenhouse gas methane. The release to the atmosphere is prevented by anaerobic 'methanotrophic archaea (ANME) dependent on a symbiotic association with sulfate-reducing bacteria or direct reduction of metal oxides. Metagenomic analyses of ANME are consistent with a reverse methanogenesis pathway, although no wild-type isolates have been available for validation and biochemical investigation. Herein is reported the characterization of methanotrophic growth for the diverse marine methanogens Methanosarcina acetivorans C2A and Methanococcoides orientis sp. nov. Growth was dependent on reduction of either ferrihydrite or humic acids revealing a respiratory mode of energy conservation. Acetate and/or formate were end products. Reversal of the well-characterized methanogenic pathways is remarkably like the consensus pathways for uncultured ANME based on extensive metagenomic analyses.

Magnetite nanoparticle coating chemistry regulates root uptake pathways and iron chlorosis in plants.

Understanding the fundamental interaction of nanoparticles at plant interfaces is critical for reaching field-scale applications of nanotechnology-enabled plant agriculture, as the processes between nanoparticles and root interfaces such as root compartments and root exudates remain largely unclear. Here, using iron deficiency-induced plant chlorosis as an indicator phenotype, we evaluated the iron transport capacity of Fe3O4 nanoparticles coated with citrate (CA) or polyacrylic acid (PAA) in the plant rhizosphere. Both nanoparticles can be used as a regulator of plant hormones to promote root elongation, but they regulate iron deficiency in plant in distinctive ways. In acidic root exudates secreted by iron-deficient Arabidopsis thaliana, CA-coated particles released fivefold more soluble iron by binding to acidic exudates mainly through hydrogen bonds and van der Waals forces and thus, prevented iron chlorosis more effectively than PAA-coated particles. We demonstrate through roots of mutants and visualization of pH changes that acidification of root exudates primarily originates from root tips and the synergistic mode of nanoparticle uptake and transformation in different root compartments. The nanoparticles entered the roots mainly through the epidermis but were not affected by lateral roots or root hairs. Our results show that magnetic nanoparticles can be a sustainable source of iron for preventing leaf chlorosis and that nanoparticle surface coating regulates this process in distinctive ways. This information also serves as an urgently needed theoretical basis for guiding the application of nanomaterials in agriculture.

Probing the Surface Layer Modulation on Archaeal Mechanics and Adhesion at the Single-Cell Level.

Addressing the challenge of understanding how cellular interfaces dictate the mechanical resilience and adhesion of archaeal cells, this study demonstrates the role of the surface layer (S-layer) in methanogenic archaea. Using a combination of atomic force microscopy and single-cell force spectroscopy, we quantified the impact of S-layer disruption on cell morphology, mechanical properties, and adhesion capabilities. We demonstrate that the S-layer is crucial for maintaining cell morphology, where its removal induces significant cellular enlargement and deformation. Mechanical stability of the cell surface is substantially compromised upon S-layer disruption, as evidenced by decreased Young's modulus values. Adhesion experiments revealed that the S-layer primarily facilitates hydrophobic interactions, which are significantly reduced after its removal, affecting both cell-cell and cell-bubble interactions. Our findings illuminate the S-layer's fundamental role in methanogen architecture and provide a chemical understanding of archaeal cell surfaces, with implications for enhancing methane production in biotechnological applications.

Mechanism of transport and toxicity response of Chlorella sorokiniana to polystyrene nanoplastics.

The toxicity of nanoparticles to freshwater microalgae is of significant importance in maintaining the overall stability of aquatic ecosystems. However, the transport mechanism and toxicity response of microalgae towards nanoplastics (NPs) remain to be further investigated. In this study, we examined the toxicity and internalization mechanisms of polystyrene nanoplastics (PS-NPs) in the microalga Chlorella sorokiniana. The results revealed that the PS-NPs inhibited algal cells' growth and disrupted cell integrity upon contact, leading to cell shrinkage or rupture. Moreover, amino-modified PS-NPs (Nano-PS-NH2) exhibited greater toxicity to C. sorokiniana than carboxyl-modified PS-NPs (Nano-PS-COOH). Furthermore, significant inhibition of PS-NPs internalization was observed when four different endocytosis-related inhibitors were used, indicating that internalized PS-NPs can enter algal cells through endocytic pathways. More importantly, C. sorokiniana exposed to Nano-PS-NH2 responded to the reduction in carbon sources and energy resulting from the suppression of photosynthesis by regulating the metabolism of carbohydrates. These findings elucidate the effects of PS-NPs on C. sorokiniana, including their impact on cell morphology and metabolism, while shedding light on the internalization mechanisms of NPs by C. sorokiniana which deepen our understanding of the toxicity of nanoplastics on algae and provide important theoretical support for solving such aquatic ecological environment problems.

Core-Shell Au@Nanoplastics as a Quantitative Tracer to Investigate the Bioaccumulation of Nanoplastics in Freshwater Ecosystems.

Studies on the adverse effects of nanoplastics (NPs, particle diameter <1000 nm) including physical damage, oxidative stress, impaired cell signaling, altered metabolism, developmental defects, and possible genetic damage have intensified in recent years. However, the analytical detection of NPs is still a bottleneck. To overcome this bottleneck and obtain a reliable and quantitative distribution analysis in complex freshwater ecosystems, an easily applicable NP tracer to simulate their fate and behavior is needed. Here, size- and surface charge-tunable core-shell Au@Nanoplastics (Au@NPs) were synthesized to study the environmental fate of NPs in an artificial freshwater system. The Au core enables the quantitative detection of NPs, while the polystyrene shell exhibits NP properties. The Au@NPs showed excellent resistance to environmental factors (e.g., 1% hydrogen peroxide solution, simulating gastric fluid, acids, and alkalis) and high recovery rates (>80%) from seawater, lake water, sewage, waste sludge, soil, and sediment. Both positively and negatively charged NPs significantly inhibited the growth of duckweed (Lemna minor L.) but had little effect on the growth of cyanobacteria (Microcystis aeruginosa). In addition, the accumulation of positively and negatively charged NPs in cyanobacteria occurred in a concentration-dependent manner, with positively charged NPs more easily taken up by cyanobacteria. In contrast, negatively charged NPs were more readily internalized in duckweed. This study developed a model using a core-shell Au@NP tracer to study the environmental fate and behavior of NPs in various complex environmental systems.

In Situ Probing of the Intrinsic Adhesion Strength of Single Anaerobic Microbial Cells.

Probing the single-cell mechanobiology in situ is imperative for microbial processes in the medical, industrial, and agricultural realms, but it remains a challenge. Herein, we present a single-cell force microscopy method that can be used to measure microbial adhesion strength under anaerobic conditions in situ. This method integrates atomic force microscopy with an anaerobic liquid cell and inverted fluorescence microscopy. We obtained the nanomechanical measurements of the single anaerobic bacterium Ethanoligenens harbinense YUAN-3 and the methanogenic archaeon Methanosarcina acetivorans C2A and their nanoscale adhesion forces in the presence of sulfoxaflor, a successor of neonicotinoid pesticides. This study presents a new tool for in situ single-cell force measurements of various anoxic and anaerobic species and provides new perspectives for evaluating the potential environmental risk of neonicotinoid applications in ecosystems.

Degradation of Antibiotic Resistance Genes by VADER with CRISPR-Cas Immunity.

The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes, e.g., biological wastewater treatment, are key barriers to prevent the spread of ARGs, they are often sources of ARGs at the same time, requiring upgraded biotechnology. Here, we present VADER, a synthetic biology system for the degradation of ARGs based on CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, to be implemented for wastewater treatment processes. Navigated by programmable guide RNAs, VADER targets and degrades ARGs depending on their DNA sequences, and by employing an artificial conjugation machinery, IncP, it can be delivered via conjugation. The system was evaluated by degrading plasmid-borne ARGs in Escherichia coli and further demonstrated via the elimination of ARGs on the environmentally relevant RP4 plasmid in Pseudomonas aeruginosa. Next, a prototype conjugation reactor at a 10-mL scale was devised, and 100% of the target ARG was eliminated in the transconjugants receiving VADER, giving a proof of principle for the implementation of VADER in bioprocesses. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. IMPORTANCE Antibiotic resistance has been causing severe health problems and has led to millions of deaths in recent years. Environmental processes, especially those of the wastewater treatment sector, are an important barrier to the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as a nonnegligible source of antibiotic resistance at the same time, as antibiotic resistance with its main cause, antibiotic resistance genes (ARGs), may accumulate in biological treatment units. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to tackle the antibiotic resistance problem raised in wastewater treatment processes, and we propose a new sector specialized in ARG removal with a conjugation reactor to implement the CRISPR-Cas system. Our study provides a new angle for resolving public health issues via the implementation of synthetic biology in environmental contexts at the process level.

Bubbles Expand the Dissemination of Antibiotic Resistance in the Aquatic Environment.

Antibiotic resistance is a global health challenge, and the COVID-19 pandemic has amplified the urgency to understand its airborne transmission. The bursting of bubbles is a fundamental phenomenon in natural and industrial processes, with the potential to encapsulate or adsorb antibiotic-resistant bacteria (ARB). However, there is no evidence to date for bubble-mediated antibiotic resistance dissemination. Here, we show that bubbles can eject abundant bacteria to the air, form stable biofilms over the air-water interface, and provide opportunities for cell-cell contact that facilitates horizontal gene transfer at and over the air-liquid interface. The extracellular matrix (ECM) on bacteria can increase bubble attachment on biofilms, increase bubble lifetime, and, thus, produce abundant small droplets. We show through single-bubble probe atomic force microscopy and molecular dynamics simulations that hydrophobic interactions with polysaccharides control how the bubble interacts with the ECM. These results highlight the importance of bubbles and its physicochemical interaction with ECM in facilitating antibiotic resistance dissemination and fulfill the framework on antibiotic resistance dissemination.

Microplastics exhibit accumulation and horizontal transfer of antibiotic resistance genes.

Although the fates of microplastics (0.1-5 mm) in marine environments and freshwater are increasingly studied, little is known about their vector effect in wastewater treatment plants (WWTPs). Previous studies have evaluated the accumulation of antibiotic resistance genes (ARGs) on microplastics, but there is no direct evidence for the selection and horizontal transfer of ARGs on different microplastics in WWTPs. Here, we show biofilm formation as well as bacterial community and ARGs in these biofilms grown on four kinds of microplastics via incubation in the aerobic and anaerobic tanks of a WWTP. Microplastics showed differential capacities for bacteria and ARGs enrichment, differing from those of the culture environment. Furthermore, ARGs in microplastic biofilms were horizontally transferred at frequencies higher than those in water samples in both tanks. Therefore, microplastics in WWTPs can act as substrates for horizontal transfer of ARGs, potentially causing a great harm to the ecological environment and adversely affecting human health.

Do Polystyrene Nanoplastics Have Similar Effects on Duckweed (Lemna minor L.) at Environmentally Relevant and Observed-Effect Concentrations?

Although the biological effects of nanoplastics (<100 nm in size) in aquatic environments have been increasingly investigated, almost all such studies have been performed at observed-effect concentrations (higher than 1 µg/mL). The use of observed-effect concentrations of nanoplastics can provide essential data for evaluating the potential risks, but how these results apply to the effects of concentrations of nanoplastics observed in the environment remains unclear. Here, we show that exposure to both positively and negatively charged nanoplastics at the observed-effect concentration (ranging from 0 to 50 µg/mL) can result in physiological changes of Lemna minor L., a typical flowering aquatic plant species, inducing H2O2 and O2- accumulation and even cell death. However, the nanoplastics at environmentally relevant concentrations (lower than 0.1 µg/mL) had no obvious effects on phenotype of L. minor. Moreover, nanoplastics at both observed-effect and environmentally relevant concentrations were adsorbed onto the roots and fronds of the plants, whereas uptake by the roots and fronds occurred only at the observed-effect concentration. Although no phenotypic changes across 30 generations of cultivation were observed when the plants were exposed to 0.015 µg/mL nanoplastics, the expression of genes related to the response to stimuli and to oxidative and osmotic stress was upregulated under both observed-effect and environmentally relevant concentrations. Our findings suggest that the long-term presence of nanoplastics at environmentally relevant concentrations might induce some variations in the transcription level and have potential threat to floating microphytes and aquatic ecosystems.

Model-based mid-infrared spectroscopy for on-line monitoring of volatile fatty acids in the anaerobic digester.

The performance of anaerobic digestion is significantly governed by the concentration of volatile fatty acids (VFAs). Though the titration and near-infrared spectroscopy have been used to measure the VFAs in the digester, there is still lack of the establishment of on-line monitoring of VFAs in practical application. An effective quantification method based on mid-infrared (MIR) spectroscopy was developed, and used to measure the concentrations of VFAs in the anaerobic bioreactor nondestructively in parallel. The wavelet denoising (WD) spectra were used as the spectral preprocessing option. Compared with other pretreatment methods, the established calibration model built by WD spectra showed satisfactory results. Further, the model was verified using high performance liquid chromatography (HPLC), and predictions were made using real reactor effluent samples. Based on this theoretical work, a set of equipment for the in-situ online monitoring of VFAs was designed, which has high feasibility and effectively solves the problems with the current VFAs online monitoring process. These results provide a new solution for on-line monitoring of the anaerobic digestion, and have great potential for practical application.

Aged microplastics enhance their interaction with ciprofloxacin and joint toxicity on Escherichia coli.

Microplastics (MPs) in natural environments undergo complex aging processes, changing their interactions with coexisting antibiotics, and posing unpredictable ecological risks. However, the joint toxicity of aged MPs (aMPs) and antibiotics to bacteria, especially at the molecular level, is unclear. In this study, non-thermal plasma technology was used to simultaneously simulate various radical oxidation and physical reactions that occur naturally in the environment, breaking the limitation of simple aging process in laboratory aging technologies. After aging, we investigated the altered properties of aMPs, their interactions with ciprofloxacin (CIP), and the molecular responses of E. coli exposed to pristine MPs (13.5 mg/L), aMPs (13.5 mg/L), and CIP (2 µg/L) individually or simultaneously. aMPs bound far more CIP to their surfaces than pristine MPs, especially in freshwater ecosystems. Notably, the growth of E. coli exposed to aMPs alone was inhibited, whereas pristine MPs exposure didn't affect the growth of E. coli. Moreover, the most differentially expressed genes in E. coli were induced by the coexposure of aMPs and CIP. Although E. coli depended on chemotaxis to improve its flagellar rotation and escaped the stress of pollutants, the coexposure of aMPs and CIP still caused cell membrane damage, oxidative stress, obstruction of DNA replication, and osmotic imbalance in E. coli. This study filled the knowledge gap between the toxicity of aMPs and pristine MPs coexisting with antibiotics at the transcription level, helping in the accurate assessment of the potential risks of MPs to the environment.

CO2 is a key constituent of the plant growth-promoting volatiles generated by bacteria in a sealed system.

KEY MESSAGE: Plant growth is greatly inhibited in tightly sealed Petri dishes for lack of CO2. Bacteria which co-cultured with plant can produce CO2 to promote plant growth in sealed systems. Bacteria produce a wide variety of volatiles, some of which can support and others can damage plant growth. It is a controversial issue whether CO2 or other bacterial volatile compounds promote plant growth in sealed systems. CO2 is critical for photosynthesis. Here, we show that CO2 is a key constituent of the plant growth-promoting volatiles generated by bacteria in a sealed system. We revealed that the growth of Arabidopsis seedlings in an airtight container was retarded due to insufficient supply of the CO2. When either CO2 was introduced into the container, or the seedlings were co-cultured along with certain bacterial species, the plants' growth was restored. CONCLUSION: The benefit of co-culturing was largely due to the CO2 generated by respiration of the bacteria.

PIFs coordinate shade avoidance by inhibiting auxin repressor ARF18 and metabolic regulator QQS.

Shade avoidance syndrome (SAS) arises in densely growing plants that compete for light. In Arabidopsis thaliana, phytochrome interacting factor (PIF) proteins link the perception of shade to stem elongation via auxin production. Here, we report that PIFs inhibit the shade-induced expression of AUXIN RESPONSE FACTOR 18 (ARF18), and ARF18 represses auxin signaling. Therefore, PIF-mediated inhibition of ARF18 enhances auxin-dependent hypocotyl elongation in simulated shade. Furthermore, we show that both PIFs and ARF18 directly repress qua-quine starch (QQS), which controls the allocation of carbon and nitrogen. Shade-repressed QQS attenuates the conversion of starch to protein and thus reduced leaf area. Our results suggest that PIF-dependent gene regulation coordinates multiple SAS responses, including altered stem growth via ARF18, as well as altered leaf growth and metabolism via QQS.

Nanoplastics Promote Microcystin Synthesis and Release from Cyanobacterial Microcystis aeruginosa.

Although the fate of nanoplastics (<100 nm) in freshwater systems is increasingly well studied, much less is known about its potential threats to cyanobacterial blooms, the ultimate phenomenon of eutrophication occurrence worldwide. Previous studies have evaluated the consequences of nanoplastics increasing the membrane permeability of microbes, however, there is no direct evidence for interactions between nanoplastics and microcystin; intracellular hepatotoxins are produced by some genera of cyanobacteria. Here, we show that the amino-modified polystyrene nanoplastics (PS-NH2) promote microcystin synthesis and release from Microcystis aeruginosa, a dominant species causing cyanobacterial blooms, even without the change of coloration. We demonstrate that PS-NH2 inhibits photosystem II efficiency, reduces organic substance synthesis, and induces oxidative stress, enhancing the synthesis of microcystin. Furthermore, PS-NH2 promotes the extracellular release of microcystin from M. aeruginosa via transporter protein upregulation and impaired cell membrane integrity. Our findings propose that the presence of nanoplastics in freshwater ecosystems might enhance the threat of eutrophication to aquatic ecology and human health.

AtHB7/12 Regulate Root Growth in Response to Aluminum Stress.

Aluminum (Al) stress is a major limiting factor for plant growth and crop production in acid soils. At present, only a few transcription factors involved in the regulation of Al resistance have been characterized. Here, we used reversed genetic approach through phenotype analysis of overexpressors and mutants to demonstrate that AtHB7 and AtHB12, two HD-Zip I transcription factors, participate in Al resistance. In response to Al stress, AtHB7 and AtHB12 displayed different dynamic expression patterns. Although both AtHB7 and AtHB12 positively regulate root growth in the absence of Al stress, our results showed that AtHB7 antagonizes with AtHB12 to control root growth in response to Al stress. The athb7/12 double mutant displayed a wild-type phenotype under Al stress. Consistently, our physiological analysis showed that AtHB7 and AtHB12 oppositely regulate the capacity of cell wall to bind Al. Yeast two hybrid assays showed that AtHB7 and AtHB12 could form homo-dimers and hetero-dimers in vitro, suggesting the interaction between AtHB7 and AtHB12 in the regulation of root growth. The conclusion was that AtHB7 and AtHB12 oppositely regulate Al resistance by affecting Al accumulation in root cell wall.

Ethylene promotes cadmium-induced root growth inhibition through EIN3 controlled XTH33 and LSU1 expression in Arabidopsis.

Cadmium (Cd) stress is one of the most serious heavy metal stresses limiting plant growth and development. However, the molecular mechanisms underlying Cd-induced root growth inhibition remain unclear. Here, we found that ethylene signalling positively regulates Cd-induced root growth inhibition. Arabidopsis seedlings pretreated with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid exhibited enhanced Cd-induced root growth inhibition, whereas the addition of the ethylene biosynthesis inhibitor aminoethoxyvinyl glycine decreased Cd-induced root growth inhibition. Consistently, ethylene-insensitive mutants, such as ein4-1, ein3-1 eil1-1 double mutant, and EBF1ox, displayed an increased tolerance to Cd. Furthermore, we also observed that Cd inhibited EIN3 protein degradation, a process that was regulated by ethylene signalling. Genetic and biochemical analyses showed that EIN3 enhanced root growth inhibition under Cd stress through direct binding to the promoters and regulating the expression of XTH33 and LSU1, which encode key regulators of cell wall extension and sulfur metabolic process, respectively. Collectively, our study demonstrates that ethylene plays a positive role in Cd-regulated root growth inhibition through EIN3-mediated transcriptional regulation of XTH33 and LSU1 and provides a molecular framework for the integration of environmental signals and intrinsic regulators in modulating plant root growth.

Micelle-mediated transport disturbance providing extracellular strategy for alleviating n-butanol stress on Escherichia coli.

One barrier inhibiting further progress in biofuel production is the toxicity of biofuels towards their producers. It is promising to apply gene-based intracellular techniques to engineer better strains with higher organic solvent tolerance. These methods are, however, complex. In the present study, we developed a simple, manageable, and commercial extracellular prototypal strategy to alleviate n-butanol (n-BuOH) stress on Escherichia coli via a micelle-mediated transport disturbance. When the concentration of sodium dodecyl sulfate, a typical anionic surfactant, is high enough to form micelles, n-BuOH will be trapped into/onto the micelles, and the negative charge prevents the n-BuOH from approaching the cells. Our study provides an extracellular strategy to relieve the stress from n-BuOH, and it also exhibits a new angle to advance microbial factories through extracellular routines.