Engineering bionanoreactor in bacteria for efficient hydrogen production.

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 µmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.

Comprehensive Techno-environmental Evaluation of a Pilot-Scale PHA Production from Food Waste in China.

Polyhydroxyalkanoates (PHAs), a biodegradable plastic that might replace petroleum-based plastics, can be recovered from organic waste using mixed microbial cultures (MMCs). Research in this field has been ongoing for about 25 years and is now in a critical commercialization period. However, few pilot-scale studies are available to analyze its technical feasibility and environmental impact. We ran an MMC PHA production pilot plant for 6 months using local food waste as the feedstock. The traditional three-stage process achieved PHA content of 47.91 ± 1.91% dry cell weight and volumetric productivity of 9.94 ± 0.01 g/L·d, while a novel rapid proliferation stage was built in, the PHA content and productivity could reach 41.39 ± 2.39% cell dry weight and 20.02 ± 0.01 g/L·d, respectively. Life cycle assessment using field data showed that greenhouse warming potential was much more than five times that of the known literature, and the fossil depletion potential was 10.30 (scenario #1)/7.59 (scenario #2) times higher than petroleum-based polyethylene (PE) plastic. However, establishing a resource-energy-water union instead of an isolated plant could achieve environmental benefits compared to PE plastic. This techno-environmental analysis provides emerging MMC PHA producers worldwide with a valuable reference for further development opportunities and market planning.

Plant Cell Wall-Penetrable, Redox-Responsive Silica Nanoprobe for the Imaging of Starvation-Induced Vesicle Trafficking.

Autophagy is a self-protection process against reactive oxygen species (ROS). The intracellular level of ROS increased when cells were cultured under nutrient starvation. Antioxidants such as glutathione and ascorbic acid play an important role in ROS removal. However, the cellular redox state in the autophagic pathway is still unclear. Herein, we developed a new redox-sensitive probe with a disulfide-linked silica scaffold to enable the sensing of the reduction environment in cell organelles. This redox-responsive silica nanoprobe (ReSiN) could penetrate the plant cell wall and release fluorescent molecules in response to redox states. By applying the ReSiN to tobacco BY-2 cells and tracing the distribution of fluorescence, we found a higher reducing potential in the central vacuole than in the autolysosomes. Upon cysteine protease inhibitor (E64-c) treatment in sucrose-free medium, the disulfide-silica structures of the ReSiNs were broken down in the vacuoles but were not degraded and were accumulated in the autolysosomes. These results reveal the feasibility of our nanoprobe for monitoring the endocytic and macroautophagic pathways. These pathways merge upstream of the central vacuole, which is the final destination of both pathways. In addition, different redox potentials were observed in the autophagic pathway. Finally, the expression of the autophagy-related protein (Atg8) fused with green fluorescence protein confirmed that the ReSiN treatment itself did not induce the autophagic pathway under normal physiological conditions, indicating the versatility of this nanoprobe in studying stimuli-triggered autophagy-related trafficking.

Rhodopsin-based light-harvesting system for sustainable synthetic biology.

Rhodopsins, a diverse class of light-sensitive proteins found in various life domains, have attracted considerable interest for their potential applications in sustainable synthetic biology. These proteins exhibit remarkable photochemical properties, undergoing conformational changes upon light absorption that drive a variety of biological processes. Exploiting rhodopsin's natural properties could pave the way for creating sustainable and energy-efficient technologies. Rhodopsin-based light-harvesting systems offer innovative solutions to a few key challenges in sustainable engineering, from bioproduction to renewable energy conversion. In this opinion article, we explore the recent advancements and future possibilities of employing rhodopsins for sustainable engineering, underscoring the transformative potential of these biomolecules.

S-nitrosocysteamine-functionalised porous graphene oxide nanosheets as nitric oxide delivery vehicles for cardiovascular applications.

Nitric oxide (NO) is a key signalling molecule released by vascular endothelial cells that is essential for vascular health. Low NO bioactivity is associated with cardiovascular diseases, such as hypertension, atherosclerosis, and heart failure and NO donors are a mainstay of drug treatment. However, many NO donors are associated with the development of tolerance and adverse effects, so new formulations for controlled and targeted release of NO would be advantageous. Herein, we describe the design and characterisation of a novel NO delivery system via the reaction of acidified sodium nitrite with thiol groups that had been introduced by cysteamine conjugation to porous graphene oxide nanosheets, thereby generating S-nitrosated nanosheets. An NO electrode, ozone-based chemiluminescence and electron paramagnetic resonance spectroscopy were used to measure NO released from various graphene formulations, which was sustained at >5 × 10-10 mol cm-2 min-1 for at least 3 h, compared with healthy endothelium (cf. 0.5-4 × 10-10 mol cm-2 min-1). Single cell Raman micro-spectroscopy showed that vascular endothelial and smooth muscle cells (SMCs) took up graphene nanostructures, with intracellular NO release detected via a fluorescent NO-specific probe. Functionalised graphene had a dose-dependent effect to promote proliferation in endothelial cells and to inhibit growth in SMCs, which was associated with cGMP release indicating intracellular activation of canonical NO signalling. Chemiluminescence detected negligible production of toxic N-nitrosamines. Our findings demonstrate the utility of porous graphene oxide as a NO delivery vehicle to release physiologically relevant amounts of NO in vitro, thereby highlighting the potential of these formulations as a strategy for the treatment of cardiovascular diseases.

Engineering artificial photosynthesis based on rhodopsin for CO2 fixation.

Microbial rhodopsin, a significant contributor to sustaining life through light harvesting, holds untapped potential for carbon fixation. Here, we construct an artificial photosynthesis system which combines the proton-pumping ability of rhodopsin with an extracellular electron uptake mechanism, establishing a pathway to drive photoelectrosynthetic CO2 fixation by Ralstonia eutropha (also known as Cupriavidus necator) H16, a facultatively chemolithoautotrophic soil bacterium. R. eutropha is engineered to heterologously express an extracellular electron transfer pathway of Shewanella oneidensis MR-1 and Gloeobacter rhodopsin (GR). Employing GR and the outer-membrane conduit MtrCAB from S. oneidensis, extracellular electrons and GR-driven proton motive force are integrated into R. eutropha's native electron transport chain (ETC). Inspired by natural photosynthesis, the photoelectrochemical system splits water to supply electrons to R. eutropha via the Mtr outer-membrane route. The light-activated proton pump - GR, supported by canthaxanthin as an antenna, powers ATP synthesis and reverses the ETC to regenerate NADH/NADPH, facilitating R. eutropha's biomass synthesis from CO2. Overexpression of a carbonic anhydrase further enhances CO2 fixation. This artificial photosynthesis system has the potential to advance the development of efficient photosynthesis, redefining our understanding of the ecological role of microbial rhodopsins in nature.

Deep insight into oriented propionate production from food waste: Microbiological interpretation and design practice.

Using mixed microbial cultures (MMCs) for oriented volatile fatty acids (VFAs) refining in an open environment is a typical challenge due to the microbial diversiform and the process complexity. Especially for carbohydrate-rich waste (such as food waste), butyrate-type fermentation is usually dominant in a single-stage MMCs anaerobic process, while the production of odd-carbon VFAs (such as propionate) is difficult although it plays a significant role in chemicals industries. In this study, firstly, we gave a new perspective on the rationality of the oriented propionate production using MMCs with lactate as feedstock by conducting in-depth microbial informatics and reaction analysis. Secondly, we verified the feasibility of the "food waste-lactate-propionate" route to reverse the original butyrate-type fermentation situation and explore mechanisms for maintaining stability. In the first stage, a defined lactate fermentation microbiome was used to produce lactate-containing broth (80% of total chemical oxygen demand) at pH=4. In the second stage, an undomesticated undefined anaerobic microbiome was used to drive propionate production (45.26% ± 2.23% of total VFAs) under optimized conditions (C/N = 100:1-200:1 and pH=5.0). The low pH environment in the first stage enhanced the lactic acid bacteria to resist the invasion of non-functional flanking bacteria, making the community stable. In the second stage, the system maintained the propionate-type fermentation due to the absence of the ecological niche of the invasive lactic acid bacteria; The selection of propionate-producing specialists was a necessary but not sufficient condition for propionate-type fermentation. At last, this study proposed an enhanced engineering strategy framework for understanding elaborate MMCs fermentation.

Artificial intelligence-aided rapid and accurate identification of clinical fungal infections by single-cell Raman spectroscopy.

Integrating artificial intelligence and new diagnostic platforms into routine clinical microbiology laboratory procedures has grown increasingly intriguing, holding promises of reducing turnaround time and cost and maximizing efficiency. At least one billion people are suffering from fungal infections, leading to over 1.6 million mortality every year. Despite the increasing demand for fungal diagnosis, current approaches suffer from manual bias, long cultivation time (from days to months), and low sensitivity (only 50% produce positive fungal cultures). Delayed and inaccurate treatments consequently lead to higher hospital costs, mobility and mortality rates. Here, we developed single-cell Raman spectroscopy and artificial intelligence to achieve rapid identification of infectious fungi. The classification between fungi and bacteria infections was initially achieved with 100% sensitivity and specificity using single-cell Raman spectra (SCRS). Then, we constructed a Raman dataset from clinical fungal isolates obtained from 94 patients, consisting of 115,129 SCRS. By training a classification model with an optimized clinical feedback loop, just 5 cells per patient (acquisition time 2 s per cell) made the most accurate classification. This protocol has achieved 100% accuracies for fungal identification at the species level. This protocol was transformed to assessing clinical samples of urinary tract infection, obtaining the correct diagnosis from raw sample-to-result within 1 h.

Deciphering the extracellular and intracellular antibiotic resistance genes in multiple environments reveals the persistence of extracellular ones.

The extracellular and intracellular antibiotic resistance genes (eARGs and iARGs) together constitute the entire resistome in environments. However, the systematic analysis of eARGs and iARGs was still inadequate. Three kinds of environments, i.e., livestock manure, sewage sludge, and lake sediment, were analyzed to reveal the comprehensive characteristics of eARGs and iARGs. Based on the metagenomic data, the diversities, relative abundances, and compositions of eARGs and iARGs were similar. The extracellular and intracellular integrons and insertion sequences (ISs) also did not show any significant differences. However, the degree and significance of the correlation between total relative abundances of integrons/ISs and ARGs were lower outside than inside the cells. Gene cassettes carried by class 1 integron were amplified in manure and sludge samples, and sequencing results showed that the identified ARGs extracellularly and intracellularly were distinct. By analyzing the genetic contexts, most ARGs were found located on chromosomes. Nevertheless, the proportion of ARGs carried by plasmids increased extracellularly. qPCR was employed to quantify the absolute abundances of sul1, sul2, tetO, and tetW, and their extracellular proportions were found highest in sludge samples. These findings together raised the requirements of considering eARGs and iARGs separately in terms of risk evaluation and removal management.

Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO2 Fixation.

A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO2 to biomass, chemicals, and fuels. We engineered a light-dependent electron transfer chain by integrating rhodopsin and an electron donor to form a closed redox loop, which drives rhodopsin-dependent CO2 fixation. A light-driven proton pump comprising Gloeobacter rhodopsin (GR) and its cofactor retinal have been assembled in Ralstonia eutropha (Cupriavidus necator) H16. In the presence of light, this strain fixed inorganic carbon (or bicarbonate) leading to 20% growth enhancement, when formate was used as an electron donor. We found that an electrode from a solar panel can replace organic compounds to serve as the electron donor, mediated by the electron shuttle molecule riboflavin. In this new autotrophic and photo-electrosynthetic system, GR is augmented by an external photocell for reductive CO2 fixation. We demonstrated that this hybrid photo-electrosynthetic pathway can drive the engineered R. eutropha strain to grow using CO2 as the sole carbon source. In this system, a bioreactor with only two inputs, light and CO2, enables the R. eutropha strain to perform a rhodopsin-dependent autotrophic growth. Light energy alone, supplied by a solar panel, can drive the conversion of CO2 into biomass with a maximum electron transfer efficiency of 20%.

Pilot-scale fermentation of urban food waste for volatile fatty acids production: The importance of pH.

Here, a pilot-scale volatile fatty acids (VFAs) production system was established using food waste (FW) as feedstock under acidic conditions. The effects of pH (uncontrolled, 4.5, 5.5, and 6.5) on the FW acidification system were investigated. The results showed that VFAs concentration increased from 8419 to 15048 mg COD/L with pH level increasing from 4.5 to 6.5, and the highest VFA production yield (0.79 mgCOD/mgCOD) was obtained at a pH of 6.5. A larger proportion of butyric acid (52.9%) was observed, accompanied by a 23% decrease of acetic acid when pH was elevated to 6.5. Microbial analysis showed that Clostridium sensu stricto 1, Sporanaerobacter, and Proteiniphilum were dominant, which not only positively influence the hydrolysis and acidogenesis processes but also play an essential role in the conversion of acetic acid to butyric acid. In summary, this study provides a valuable reference for large-scale FW treatment to recover valuable resources.

Reducing the effect of non-volatile fatty acids (non-VFAs) on polyhydroxyalkanoates (PHA) production from fermented thermal-hydrolyzed sludge.

Thermal-hydrolyzed sludge (THS) can be fermented to produce volatile fatty acids (VFAs) rich liquids. These fermentative liquids are considered as a potential feedstock for polyhydroxyalkanoates (PHA) production. However, the presence of high levels of non-VFAs organics supporting the growth response instead of PHA accumulation hindered an efficient culture selection in a feast and famine regime. Lowering the non-VFAs content can compromise activities of microorganisms to take up these external carbon sources and improve the selective pressure; thus two enhanced strategies were tested to optimize the selection process: 1) increasing the proportion of VFAs in the original substrate or 2) removing most of the non-VFAs at the end of the feast phase. Results showed that the strategies resulted in PHA yields on VFAs respectively of 0.62 and 0.54 Cmol/Cmol, significantly higher than that in original SBR (0.16-0.35 Cmol/Cmol), confirming that reducing the adverse effect of non-VFAs can impose effective internal limitation and induce high PHA storage responses. In PHA accumulation tests, cultures selected with synthetic substrates accumulated a maximum PHA content of 61.4 wt%, which is the highest as ever reported among PHA production from THS. In summary, the study provided valuable references for improving PHA production from complex substrates.

Anaerobic digestion reduces extracellular antibiotic resistance genes in waste activated sludge: The effects of temperature and degradation mechanisms.

The fate of extracellular antibiotic resistance genes (eARGs) in waste activated sludge during anaerobic digestion (AD) remained unclear. The current study investigated the changes in seven eARGs (sulI, sulII, tet(A), tet(O), tet(X), blaTEM, and blaSHV) and intI1 during sludge AD at 35 °C and 55 °C. First, the extracellular DNA (eDNA) extraction method from sludge was optimized by adding sodium dodecyl sulfonate, and the eDNA recovery nearly doubled. Second, analysis via qPCR showed that eARGs ranged from 1.5% to 85.1% of the total ARGs, stressing the importance of eARGs in sludge. Besides, the abundances of all detected eARGs decreased following AD, where removal rates ranged from 22.8% to 93.9% at 35 °C and 52.7% to 96.6% at 55 °C. Further analysis showed that the removal rates of eARGs were negatively correlated with their initial abundances (P < 0.05). Last, the degradation characteristics of eARGs under AD conditions were determined. The first-order degradation rate constants for different eARGs did not vary significantly, indicating that gene sequences did not cause a removal distinction, and fitted Michaelis-Menten equation confirmed the higher eARGs degradation ability at the higher temperature. Overall, this study firstly uncovered the decrease of eARGs in sludge during AD treatment, and advanced the understanding of the positive effect of AD on eARGs dissemination control.

Longitudinal and quantitative assessment platform for concurrent analysis of anti-tumor efficacy and cardiotoxicity of nano-formulated medication in vivo.

Increasing nanomedicinal approaches have been developed to effectively inhibit tumor growth; however, critical questions such as whether a nanomedicinal approach can mitigate latent side effects are barely addressed. To this end, we established a zebrafish xenograft tumor model, combining pseudodynamic three-dimensional cardiac imaging and image analysis to enable simultaneous and quantitative determination of the change of tumor volume and cardiac function of zebrafish upon specific nanoformulation treatment. Doxorubicin (DOX), a well-known chemotherapeutic agent with cardiotoxicity, and a recently developed DOX-loaded nanocomposite were employed as two model drugs to demonstrate the effectiveness to utilize the proposed evaluation platform for rapid validation. The nanoformulation significantly mitigated DOX-associated cardiotoxicity, while retaining the efficacy of DOX in inhibiting tumor growth compared to administration of carrier-free DOX at the same dose. We anticipate that this platform possesses the potential as an efficient assessment system for nanoformulated cancer therapeutics with suspected toxicity and side effects to vital organs such as the heart.

Polyhydroxyalkanoates (PHA) production from fermented thermal-hydrolyzed sludge by mixed microbial cultures: The link between phosphorus and PHA yields.

Production of polyhydroxyalkanoates (PHA) from wastes has gained increasing attention for the related low costs and high environmental benefits. Phosphorus limitation is a potential strategy used to facilitate PHA production, yet excessive limitation was previously reported to cause negative effects. This study was the first to investigate the optimum phosphorus limitation for PHA accumulation from thermal-hydrolyzed sludge. The results showed that the maximum PHA content increased from 23 wt% to 51 wt% when phosphorus concentration was limited from 127.60 to 1.35 mg/L, indicating that a lower phosphorus concentration would promote maximum PHA accumulation. Batch tests performed with synthetic substrates (containing one specific VFA for each batch) confirmed that the effect of phosphorus content on PHA production was mainly devoted by the efficiency of the conversion of acetate to PHA. The PHA yields on acetate (YPHA/ac) were 0.68 and 0.05 Cmol/Cmol under phosphorus-limited (1 mg/L) and -excess (100 mg/L) conditions, respectively. A mathematical model was developed to describe the correlation between phosphorus concentration and YPHA/ac, which can fit the experimental data and predict the results properly. Finally, further (ammonium-) nitrogen restriction did not efficiently cause the additional improvement of PHA production under the conditions of phosphorus limitation.

Polyhydroxyalkanoates (PHA) production from fermented thermal-hydrolyzed sludge by PHA-storing denitrifiers integrating PHA accumulation with nitrate removal.

Polyhydroxyalkanoates (PHA) production from fermented thermal-hydrolyzed sludge was conducted by mixed microbial cultures (MMCs) in the study. An MMC enriched in the species Brachymonas_denitrificans (60.18%) was selected under an aerobic feast/famine regime, which is capable of denitrification and accumulating PHA. To take advantage of the PHA-storing denitrifiers, an aerobic-feast/anoxic-famine regime was applied to integrate culture selection with denitrification. The results showed that cultures enriched under the regime exhibited a PHA storage capacity with PHA yield on VFA of 0.47 gCOD/gCOD and well denitrification performance achieving nitrate removal of 98%. Moreover, the aerobic-feast/anoxic-famine regime could originate a comparable maximum PHA content to the complete aerobic feast/famine regime (49.7 wt% versus. 47.1 wt%, respectively), yet reduce aeration energy input by 79% in the culture selection process. Finally, this study investigated the accumulation of nitrite and nitrous oxide during PHA based denitrification and the feasibility of integrating the process with wastewater treatment.

Effect of thermal hydrolysis pretreatment on volatile fatty acids production in sludge acidification and subsequent polyhydroxyalkanoates production.

The study compared the differences in VFAs production between raw sludge and thermal hydrolyzed sludge (TH-sludge) at different temperature (35 °C and 55 °C) in four semi-continuous acidification reactors. Optimal VFAs yield was obtained from TH-sludge at 35 °C (0.22 gVFACOD/gVS), 44.6% higher than raw sludge at 35 °C, since the advantage of TH-sludge in SCOD solubilization overcame its disadvantage of lower carbon biodegradability. Moreover, high temperature (55 °C) was proved to aid the acidification of raw sludge by 15.7% (in YVFAs), but inhibit that of TH-sludge by 12.2%, mainly due to the suppressed microbial activities under heat. Microbial community analysis showed that TH-sludge had a larger proportion of acidogenic microbes than raw sludge, mainly attributing to the increase of Selenomonadales (37.3% vs. 3.7%); high temperature enriched thermophilic proteolytic microbes, Anaerobaculum and Coprothermobacter. Finally, optimal acidified liquid from TH-sludge at 35 °C was applied for PHAs production and achieved a competitive yield of 34.6% PHAs/DCW.

Differential in situ sensing of extra- and intracellular glutathione by a novel redox-responsive silica matrix-Au nanoprobe.

We synthesized a biothiol-sensitive nanoprobe by assembling gold nanoparticles with a novel redox-responsive silica (ReSi) matrix using dithiobis (succinimidyl propionate) and (3-aminopropyl) trimethoxysilane. Thin layer disulfide-bonded networks of the ReSi could differentially respond to extra- and intracellular glutathione in cancer cells within 30 min; furthermore, targeted cellular uptake could be monitored in situ by fluorescence recovery. Sigmoidal dose-response pattern of the nanoprobes presented in this study were attributed to the buried disulfide-linked 3D nanostructure of the ReSi nanoshell, optimized at an appropriate thickness, enabling not only buffering of small redox disturbances in the extracellular milieu but also the satisfied sensitivity for rapid redox sensing. Such a ReSi-functionalized gold nanoparticle-based nanoconjugate possesses the potential to serve as an effective intracellular drug carrier for future cancer theranostics.