Bioactive Layered Double Hydroxides for Synergistic Sonodynamic/Cuproptosis Anticancer Therapy with Elicitation of the Immune Response.

Sonodynamic therapy (SDT) has promising application prospects in tumor therapy. However, SDT does not eradicate metastatic tumors. Herein, Cu-substituted ZnAl ternary layered double hydroxide nanosheets (ZCA NSs) were developed as both sonosensitizers and copper nanocarriers for synergistic SDT/cuproptosis cancer therapy. An optimized electronic structure more conducive to the sonodynamic process was obtained from ZCA NSs via the Jahn-Teller effect induced by the introduction of Cu2+, and the synthesized ZCA NSs regulated the intricate tumor microenvironment (TME) by depleting endogenous glutathione (GSH) to amplify oxidative stress for further enhanced SDT performance. Furthermore, cuproptosis was evoked by intracellular overload of Cu2+ and amplified by SDT, leading to irreversible proteotoxicity. In vitro results showed that such synergetic SDT/cuproptosis triggered immunogenic cell death (ICD) and promoted the maturation of dendritic cells (DCs). Furthermore, the as-synthesized ZCA NS-mediated SDT/cuproptosis thoroughly eradicated the in vivo solid tumors and simultaneously elicited antitumor immunity to suppress lung and liver metastasis. Overall, this work established a nanoplatform for synergistic SDT/cuproptosis with a satisfactory antitumor immunity.

Fenton Reaction in vivo and in vitro. Possibilities and Limitations.

The review considers the problem of hydrogen peroxide decomposition and hydroxyl radical formation in the presence of iron in vivo and in vitro. Analysis of the literature data allows us to conclude that, under physiological conditions, transport of iron, carried out with the help of carrier proteins, minimizes the possibility of appearance of free iron ions in cytoplasm of the cell. Under pathological conditions, when the process of transferring an iron ion from a donor protein to an acceptor protein can be disrupted due to modifications of the carrier proteins, iron ions can enter cytosol. However, at pH values close to neutral, which is typical for cytosol, iron ions are converted into water-insoluble hydroxides. This makes it impossible to decompose hydrogen peroxide according to the mechanism of the classical Fenton reaction. A similar situation is observed in vitro, since buffers with pH close to neutral are used to simulate free radical oxidation. At the same time, iron hydroxides are able to catalyze decomposition of hydrogen peroxide with formation of a hydroxyl radical. Decomposition of hydrogen peroxide with iron hydroxides is called Fenton-like reaction. Studying the features of Fenton-like reaction in biological systems is the subject of future research.

Intranasal delivery of phenytoin loaded layered double hydroxide nanoparticles improves therapeutic effect on epileptic seizures.

Improving the efficiency of antiseizure medication entering the brain is the key to reducing its peripheral toxicity. A combination of intranasal administration and nanomedicine presents a practical approach for treating epileptic seizures via bypassing the blood-brain barrier. In this study, phenytoin (PHT) loaded layered double hydroxide nanoparticles (BSA-LDHs-PHT) were fabricated via a coprecipitation - hydrothermal method for epileptic seizure control. In this study, we expound on the preparation method and characterization of BSA-LDHs-PHT. In-vitro drug release experiment shows both rapid and continuous drug release from BSA-LDHs-PHT, which is crucial for acute seizure control and chronic epilepsy therapy. In-vivo biodistribution assays after intranasal administration indicate excellent brain targeting ability of BSA-LDHs. Compared to BSA-Cyanine5.5, BSA-LDHs-Cyanine5.5 were associated with a higher brain/peripheral ratio across all tested time points. Following intranasal delivery with small doses of BSA-LDHs-PHT, the latency of seizures in the pentylenetetrazole-induced mouse models was effectively improved. Collectively, the present study successfully designed and applied BSA-LDHs-PHT as a promising strategy for treating epileptic seizures with an enhanced therapeutic effect.

Mesoporous cobalt-manganese layered double hydroxides promote the activation of calcium sulfite for degradation and detoxification of metronidazole.

Metronidazole (MNZ), a commonly used antibiotic, poses risks to water bodies and human health due to its potential carcinogenic, mutagenic, and genotoxic effects. In this study, mesoporous cobalt-manganese layered double hydroxides (CoxMny-LDH) with abundant oxygen vacancies (Ov) were successfully synthesized using the co-precipitation method and used to activate calcium sulfite (CaSO3) with slight soluble in water for MNZ degradation. The characterization results revealed that Co2Mn-LDH had higher specific areas and exhibited good crystallinity. Co2Mn-LDH/CaSO3 exhibited the best catalytic performance under optimal conditions, achieving a remarkable MNZ degradation efficiency of up to 98.1 % in only 8 min. Quenching experiments and electron paramagnetic resonance (EPR) tests showed that SO4•- and 1O2 played pivotal roles in the MNZ degradation process by activated CaSO3, while the redox cycles of Co2+/Co3+ and Mn3+/Mn4+ on the catalyst surface accelerated electron transfer, promoting radical generation. Three MNZ degradation routes were put forward based on the density functional theory (DFT) and liquid chromatography-mass spectrometer (LC-MS) analysis. Meanwhile, the toxicity analysis result demonstrated that the toxicity of intermediates post-catalytic reaction was decreased. Furthermore, the Co2Mn-LDH/CaSO3 system displayed excellent stability, reusability, and anti-interference capability, and achieved a comparably high removal efficiency across various organic pollutant water bodies. This study provides valuable insights into the development and optimization of effective heterogeneous catalysts for treating antibiotic-contaminated wastewater.

Perovskite hydroxide-based laccase mimics with controllable activity for environmental remediation and biosensing.

Constructing relatively inexpensive nanomaterials to simulate the catalytic performance of laccase is of great significance in recent years. Although research on improving laccase-like activity by regulating ligands of copper (amino acids or small organic molecules, etc.) have achieved remarkable success. There are few reports on improving laccase-like activity by adjusting the composition of metal Cu. Here, we used perovskite hydroxide AB(OH)6 as a model to evaluate the relationship between Cu based alloys and their laccase-like activity. We found that when the Cu/Mn alloy ratio of the perovskite hydroxide A point is greater than 1, the laccase-like activity of the binary alloy perovskite hydroxide is higher than that of the corresponding single Cu. Based on the measurements of XPS and ICP-MS, we deduced that the improvements of laccase-like activity mainly attribute to the ratio of Cu+/Cu2+and the content of Cu. Moreover, two types of substrates (toxic pollutants and catechol neurotransmitters) were used to successfully demonstrated such nanozymes' excellent environmental protecting function and biosensing property. This work will provide a novel approach for the construction and application of laccase-like nanozymes in the future.

Examination of Oral Candida Infection in Primary Sjögren's Syndrome Patients

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due to the hyposalivation experienced by pSS patients, oral dysbacteriosis often occurs. A common complication of pSS is the oral Candida infection. In this article, the authors describe systematic methods that can effectively diagnose oral Candida infection and identify the Candida strains using saliva, oral mucosal swabs, or mouthwash from pSS patients. The Sabouraud's Dextrose Agar (SDA), hyphal formation assay, potassium hydroxide (KOH) smear test, and calcofluor white (CFW) staining assay are used for the diagnosis of oral Candida infection. A Candida diagnostic agar is used for the identification of Candida strains. Finally, antifungal susceptibility testing is used to determine appropriate antifungal drug treatment. This standardized method can enhance the diagnosis, treatment, and future research of pSS-related oral Candida infections. Early diagnosis, using this method, can also prevent any complications arising due to delay in receiving appropriate treatment.

Advances of Layered Double Hydroxide-Based Materials for Tumor Imaging and Therapy.

Layered double hydroxides (LDH) are a class of functional anionic clays that typically consist of orthorhombic arrays of metal hydroxides with anions sandwiched between the layers. Due to their unique properties, including high chemical stability, good biocompatibility, controlled drug loading, and enhanced drug bioavailability, LDHs have many potential applications in the medical field. Especially in the fields of bioimaging and tumor therapy. This paper reviews the research progress of LDHs and their nanocomposites in the field of tumor imaging and therapy. First, the structure and advantages of LDH are discussed. Then, several commonly used methods for the preparation of LDH are presented, including co-precipitation, hydrothermal and ion exchange methods. Subsequently, recent advances in layered hydroxides and their nanocomposites for cancer imaging and therapy are highlighted. Finally, based on current research, we summaries the prospects and challenges of layered hydroxides and nanocomposites for cancer diagnosis and therapy.

Layer-by-layer growth of Cu3(HHTP)2 films on Cu(OH)2 nanowire arrays for high performance ascorbic acid sensing.

Growing three-dimensional (3D) metal organic frameworks (MOFs) via heterogeneous epitaxial growth on metal hydroxide arrays are effective for constructing electrochemical sensor. However, the growth of MOFs is difficult to control, resulting in thick and irregular morphologies and even damage the metal hydroxide template. In this work, Cu3(HHTP)2 (HHTP = 2, 3, 6, 7, 10, 11-hexahydroxytriphenylene) films with controllable thickness and morphology were successfully prepared on Cu(OH)2 nanowire arrays (NWAs) through layer-by-layer (LBL) growth method. We have discovered that the LBL cycle and the reaction solvent composition are crucial for growing homogenous MOF thin films. The Cu3(HHTP)2 based ascorbic acid (AA) sensor, fabricated in ethanol within 10 LBL cycles, generated an ultrahigh sensitivity of 821.64 µA mM-1 cm-2 in the range of 6-981.41 µM, a low detection limit of 60 nM as well as the great selectivity, stability and reproducibility. Moreover, the relative deviation for AA detection in two fruit juices were 3.22 % and 3.71 %, and the test result for human sweat fall within the normal AA concentration range, verifying the feasibility of as-prepared sensor for practical application.

MOF-derived NiCo hydroxide for highly efficient non-enzymatic glucose biosensing.

An efficient and robust electrocatalyst is significant for glucose biosensing. The emergence of metal-organic framework (MOF) derived materials opens up new avenues for the development of high-performance glucose sensing catalysts. Herein, MOF derived nickel-cobalt hydroxide supported on conductive copper sheet (NiCo-OH/Cu sheet) is prepared at room temperature. The as-obtained NiCo-OH is endowed with three-dimensional network structure which enables the effective exposure of active materials, sufficient contact between glucose molecule and catalyst. The NiCo-OH/Cu sheet is revealed as good glucose electrochemical sensing material with a wide linear range of 0.05∼6.0 mM and a high sensitivity of 1340µA mM-1cm-2. Additionally, the as-fabricated NiCo-OH/Cu sheet displays good anti-interference ability and long-term stability.

MnFe layered double hydroxides confined MnOx for peroxymonosulfate activation: A novel manner for the selective production of singlet oxygen.

Singlet oxygen (1O2) is a reactive species for the selective degradation of stubborn organic pollutants. Given its resistance to harsh water environment, the effective and exclusive generation of 1O2 is acknowledged as a key strategy to mitigate water production costs and ensure water supply safety. Herein, we synthesized MnOx intercalated MnFe layered double hydroxides (MF-MnOx) to selectively produce 1O2 through the activation of PMS. The distinctive confined structure endowed MF-MnOx with a special pathway for the PMS activation. The direct oxidation of BPA on the intercalated MnOx induced the charge imbalance in the MnFe-LDH layer, resulting in the selective generation of 1O2. Moreover, acceptable activity deterioration of MF-MnOx was observed in a 10 h continuous degradation test in actual water, substantiating the application potential of MF-MnOx. This work presents a novel catalyst for the selective production of 1O2, and evaluates its prospects in the remediation of micro-polluted water.

Preparation of highly adsorptive biochar by sequential iron impregnation under refluxing and pyrolysis at low temperature for removal of tetracycline.

Iron-doping modification is a prevailing approach for improving adsorption capability of biochar with environmental friendliness, but usually requires high temperature and suffers from iron aggregation. Herein, a highly adsorptive biochar was manufactured via sequential disperse impregnation of iron by refluxing and pyrolysis at low temperature for eliminating tetracycline (TC) from aqueous solution. Iron oxides and hydroxides were impregnated and stably dispersed on the carbon matrix as pyrolyzed at 200 °C, meanwhile abundant oxygen and nitrogen functional groups were generated on surface. The iron-doped biochar exhibited up to 891.37 mg/g adsorption capacity at pH 5, and could be recycled with high adsorption capability. The adsorption of TC should be mostly contributed to the hydrogen bonding of N/O functional groups and the hydrogen bonding/coordination of iron oxides/hydroxides. This would provide a valuable guide for dispersedly doping iron and conserving functional groups on biochar, and a super iron-doped biochar was prepared with superior recyclability.

Understanding seasonal variations in As and Pb river fluxes and their regulatory mechanisms through monitoring data.

The Doce River Basin (DRB) suffers with the adverse impacts of mining activities, due to its high level of urbanization and numerous industrial operations. In this study, we present novel insights into contaminant flow dynamics, seasonal variations, and the primary factors driving concentration levels within the region. We conducted an extensive analysis using a database sourced from the literature, which contained data on the contamination of arsenic (As) and lead (Pb) in the Doce River. Our primary aim was to investigate the patterns of As and Pb flow throughout the entire basin, their response to seasonal fluctuations, and the key parameters influencing their concentration levels. The results showed significant seasonal fluctuations in As and Pb fluxes, peaking during the rainy season. The 2015 Fundão dam breach in the DRB led to notable changes, elevating elemental concentrations, particularly As and Pb, which were subsequently transported to the Atlantic Ocean. These increased concentrations were primarily associated with iron and manganese oxides, hydroxides, and sulfates, rather than precipitation, as evidenced by regressions with low R2 values for both As (R2 = 0.07) and Pb (R2 < 0.001), concerning precipitation. The PCA analysis further supports the connection between these elements and the oxides and hydroxides of Fe and Mn. The approach employed in this study has proven to be highly effective in comprehending biogeochemical phenomena by leveraging data from the literature and could be a model for optimizing resources by capitalizing on existing information to provide valuable insights for drainage basin management, particularly during crises.

Using CuMgFe layered double oxide to replace laccase as a catalyst for abiotic humification.

Humification offers a promising avenue for sequestering dissolved organic carbon while facilitating environmental cleanup. In this study, CuMgFe layered double oxides (LDO) were applied as a catalyst to replace conventional enzymes, such as laccase, thereby enhancing the in vitro polyphenol-Maillard humification reaction. CuMgFe LDO was synthesized through calcination of CuMgFe layered double hydroxides (LDH) at 500 °C for 5 h. A suite of characterization methods confirmed the successful formation into mixed oxides (Cu2O, CuO, MgO, FeO, and Fe2O3) after thermal treatment. A rapid humification reaction was observed with CuMgFe LDO, occurring within a two-week span, likely due to a distinct synergy between copper and iron elements. Subsequent analyses identified that MgO in CuMgFe LDO also played a pivotal role in humification by stabilizing the pH of the reaction. In the absence of magnesium, LDO's humification activity was more pronounced in the early stages of the reaction, but it rapidly diminished as the reaction progressed. The efficiency of CuMgFe LDO was heightened at elevated temperatures (35 °C), while light conditions manifested a discernible effect, with a modest decrease in humification efficacy under indoor light exposure. CuMgFe LDO surpassed both laccase and MgFe LDH in performance, boasting a superior humification efficiency relative to its precursor, CuMgFe LDH. The catalysts' humification activity was modulated by their crystallinity and valence dynamics. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) results suggested that introducing the amino acid, glycine, expedited the CuMgFe LDO-fueled humification, enhancing the formation of C-N and C-C bonds in the resultant products. The humic-like substances derived from the catalyst-enhanced reaction displayed an elevated presence of aromatic configurations and a richer array of oxygen functional groups in comparison to a typical commercial humic material.

Edge interface microenvironment regulation of CoOOH/commercial activated carbon nano-hybrids enabling PMS activation for degrading ciprofloxacin.

Peroxymonosulfate (PMS) is widely employed to generate oxygen-containing reactive species for ciprofloxacin (CIP) degradation. Herein, cobalt oxyhydroxide @activated carbon (CoOOH@AC) was synthesized via a wet chemical sedimentation method to activate PMS for degradation of CIP. The result suggested AC can support the vertical growth of CoOOH nanosheets to expose high-activity Co-contained edges, possessing efficient PMS activation and degradation activity and catalytic stability. In the presence of 3.0 mg of optimal CoOOH@AC and 2 mM PMS, 96.8 % of CIP was degraded within 10 min, approximately 11.6 and 9.97 times greater than those of CoOOH/PMS and AC/PMS systems. Notably, it was disclosed that the optimal CoOOH@AC/PMS system still exhibited efficient catalytic performance in a wide pH range, different organics and common co-existing ions. Quenching experiments and electron paramagnetic resonance indicated that both radical and non-radical processes contributed to the degradation of CIP, with 1O2 and direct electron transfer accounting for the non-radical pathway and SO4•- and •OH serving as the main radical active species. Finally, possible CIP degradation pathways were proposed based on high-performance liquid chromatography-mass spectrometry. This study provided an alternate method for wastewater treatment based on PMS catalyzed by cobalt-based hydroxide.

A Cancer Nanovaccine Based on an FeAl-Layered Double Hydroxide Framework for Reactive Oxygen Species-Augmented Metalloimmunotherapy.

The complexity and heterogeneity of individual tumors have hindered the efficacy of existing therapeutic cancer vaccines, sparking intensive interest in the development of more effective in situ vaccines. Herein, we introduce a cancer nanovaccine for reactive oxygen species-augmented metalloimmunotherapy in which FeAl-layered double hydroxide (LDH) is used as a delivery vehicle with dihydroartemisinin (DHA) as cargo. The LDH framework is acid-labile and can be degraded in the tumor microenvironment, releasing iron ions, aluminum ions, and DHA. The iron ions contribute to aggravated intratumoral oxidative stress injury by the synergistic Fenton reaction and DHA activation, causing apoptosis, ferroptosis, and immunogenic cell death in cancer cells. The subsequently released tumor-associated antigens with the aluminum adjuvant form a cancer nanovaccine to generate robust and long-term immune responses against cancer recurrence and metastasis. Moreover, Fe ion-enabled T1-weighted magnetic resonance imaging can facilitate real-time tumor therapy monitoring. This cancer-nanovaccine-mediated metalloimmunotherapy strategy has the potential for revolutionizing the precision immunotherapy landscape.

Application of CuFeLDH-NCNT in ultrasound-assisted-Fenton-like process for removing ciprofloxacin from aqueous solutions.

In this study, the mesoporous material NCNT was prepared by treating carbon nanotubes (CNT) with hydrazine and subsequently loaded with Cu-Fe layered double hydroxide (CuFeLDH) to create a multiphase catalyst (CuFeLDH-NCNT). Its application as a multiphase catalyst was investigated in an ultrasound-assisted Fenton process for ciprofloxacin (CIP) degradation in aqueous solution. In addition, the impacts of catalyst dosage, ultrasonic power, H2O2 dosage, and beginning pH on CIP removal efficiency were carefully evaluated to maximize the removal efficiency of CIP. The findings indicated that the elimination rate of the initial CIP concentration of 20 mg/L surpassed 94.66% after a mere 100 min, while the TOC degradation rate was 70.4%. The high removal rate was due to the synergistic action between the nanoparticles, H2O2, and ultrasonography. The degradation intermediates of CIP were examined, and putative degradation pathways and mechanisms were postulated.

Robust anion exchange membranes based on ionic liquid grafted chitosan/polyvinyl alcohol/quaternary ammonium functionalized silica for polymer electrolyte membrane fuel cells.

In this study, 1-bromohexyl-1methylpiperidinium bromide (Br-6-MPRD) ionic liquid grafted quaternized chitosan (QCS) and polyvinyl alcohol (PVA) blends were composited with glycidyl trimethyl ammonium chloride (GTMAC) quaternized silica (QSiO2) at different dosages. Glutaraldehyde (GA) crosslinked the membranes and then processed into hydroxide form with an aqueous potassium hydroxide solution. The resultant IL-QCS/PVA/QSiO2 membranes exhibit significantly improved ionic conductivity, moderate water absorption and swelling ratio compared with the pristine IL-QCS/PVA anion exchange membrane (AEM). Among them, the hydroxide ion conductivity and power density of IL-QCS/PVA/QSiO2-7 membrane can reach up to 78 mS cm-1 at 80 °C and 115 mW cm-2 at 60 °C respectively. In addition, IL-QCS/PVA/QSiO2 membranes have excellent thermal, mechanical, and chemical stabilities, which can meet the application requirements of AEM for fuel cells.

Performance and mechanism of biochar@FeMg-LDH for efficient activation of persulfate for degradation of 2, 4-dichlorophenol in groundwater.

Groundwater environments are complex, and traditional advanced oxidation technologies mainly based on free radicals have limitations such as poor selectivity and low interference resistance, making it difficult to efficiently degrade target pollutants in groundwater. Therefore, we developed a sludge-based biochar-supported FeMg-layered double hydroxide catalyst (BC@FeMg-LDH) for the catalytic degradation of 2, 4-dichlorophenol (2, 4-DCP) using persulfate (PDS) as an oxidant. The removal efficiency of the catalyst exceeded 95%, showing high oxidation activity in a wide pH range while being almost unaffected by reducing substances and ions in the environment. Meanwhile, under neutral conditions, the leaching of metal ions from BC@FeMg-LDH was minimal, thereby eliminating the risk of secondary pollution. According to quenching experiments and electron paramagnetic resonance spectroscopy, the main active species during BC@FeMg-LDH/PDS degradation of 2, 4-DCP is 1O2, indicating a non-radical reaction mechanism dominated by 1O2. Characterization techniques, including X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy, revealed that the carbonyl (C = O) and metal hydroxyl (M-OH) groups on the material surface were the main reactive sites mediating 1O2 generation. The 1O2 generation mechanism during the reaction involved ketone-like activation of carbonyl groups on the biochar surface and complexation of hydroxyl groups on the material surface with PDS, resulting in the formation of O2·- and further generation of 1O2. 1O2 exhibited high selectivity toward electron-rich organic compounds such as 2, 4-DCP and demonstrated strong interference resistance in complex groundwater environments. Therefore, BC@FeMg-LDH holds promising applications for the remediation of organic-contaminated groundwater.

Preparation of Oily Sludge-Derived Activated Carbon and Its Adsorption Performance for Tetracycline Hydrochloride.

Oily sludge-derived activated carbon was prepared using the potassium hydroxide (KOH) activation method using oily sludge as a raw material, and one-factor experiments determined the best conditions for preparing activated carbon. The activated carbon's morphological structure and surface chemical properties were analyzed by scanning different characterization tools, and the adsorption behavior of tetracycline hydrochloride was investigated. The results showed that the optimum conditions for preparing oily sludge-derived activated carbon were an activation temperature of 400 °C, activation time of 30 min, activator concentration of 1 mol/L, and impregnation ratio of 2 mL/g. After activation, the activated carbon had more pores and a more orderly crystal structure arrangement, the specific surface area was 2.07 times higher than that before activation, and the surface was rich in functional groups such as -HO, -C-O, -C=C, and -C-H, which increased the active sites of activated carbon. Physicochemical effects dominated the adsorption process. It belonged to the spontaneous heat absorption process under the quasi-secondary kinetic and Langmuir isothermal models. The maximum monolayer adsorption capacity of KOH-activated carbon was 205.1 mg·g-1.

New insights into biochar ammoniacal nitrogen adsorption and its correlation to aerobic degradation ammonia emissions.

One of the technical barriers to the wider use of biochar in the composting practices is the lack of accurate quantification linking biochar properties to application outcomes. To address this issue, this paper investigates the use of ammonia nitrogen adsorption capacity by biochar as a predictor of ammonia emission during composting in the presence of biochar. With this in mind, this work investigated the use of ammonia nitrogen adsorption capacity of biochar when mixed with solid digestate, and the reduction in ammonia emissions resulting from the addition of biochar during aerobic degradation of solid digestate. A biochar synthesized at 900 °C, another synthesized at 450 °C, and two derivatives of the latter biochar, one chemically modified with nitric acid and the other with potassium hydroxide, were tested. This study concluded that the chemical characteristics of the biochar, including pH and oxygen/carbon atomic ratio, had a greater influence on the adsorption of ammonia nitrogen than physical attributes such as specific surface area. In this regard, nitric acid modification had superior performance compared to hydroxide potassium modification to increase biochar chemical attributes and reduce ammonia emissions when applied to aerobic degradation. Finally, a significant linear correlation (p-value < 0.05, r2 = 0.79) was found between biochar ammonia nitrogen adsorption capacity and ammonia emissions along composting, showing the potential of this variable as a predictive parameter. This study provides insights for future explorations aiming to develop predictive tests for biochar performance.