Effect of photocatalysis on the physicochemical properties of liquid digestate.

Antibiotic residues pose a risk to the agricultural application of liquid digestate. In our previous study, photocatalysis was employed to degrade the antibiotics in liquid digestate and observed that the removal efficiency of TC, OTC, and CTC was up to 94.99%, 88.92%, and 95.52%, respectively, at the optimal experimental level, demonstrating the feasibility of this technology. In this study, the liquid digestate after photocatalysis was analyzed to evaluate the effect of photocatalysis on the nutrients, phytotoxicity, and bacterial community of liquid digestate. The results showed that photocatalysis had little effect on the major nutrients TN, TP, and TK in liquid digestate. However, photocatalysis could cause an increase in tryptophan substances as well as soluble microbial by-products and a decrease in humic acid substances in the liquid digestate. The toxicity of liquid digestate after photocatalysis exhibited an increasing trend followed by a decreasing trend, and the liquid digestate after photocatalysis for 2 h had a promoting effect on seed germination and root growth. The richness, diversity, and evenness of bacterial communities in liquid digestate were decreased as a result of photocatalysis. The dominant species in the liquid digestate was dramatically changed by photocatalysis, and the antibiotic concentration also had a major effect on the dominant species in the liquid digestate after photocatalysis. After photocatalysis for 2 h, the dominant species in the liquid digestate changed from Firmicutes to Proteobacteria.

Effects of different fermentation synergistic chemical treatments on the performance of wheat straw as a nursery substrate.

Wheat straw is rich in organic matter and nutrients and has the potential to replace peat as the primary raw material in organic nurseries. Using straw as a peat substitute can also aid in reducing the CO2 emissions that result from peat mining. Furthermore, this can avoid resource wastage and eliminate the practice of burning wheat straw, thereby causing pollution. The conventional composting treatment has a long cycle and inability to control substrate properties in a targeted manner. Thus, this study analyzed the physicochemical properties, material science properties, and biological toxicity of straw substrate at the end of fermentation to achieve rapid and targeted regulations of the substrate's overall performance. Wheat straw treated with two types of fermentation (aerobic/anaerobic) and five chemical conditioners (1% CH3COOH, 1% H2SO4, 1% NaOH, 1% K2CO3, and H2O) under different temperature conditions was used. Adjusting the pH of straw substrate to acidic levels (4.47-6.51) reduced the organic matter consumption by 0.27-5.82% under anaerobic digestion than under aerobic composting. Meanwhile, aerobic composting retained more nitrogen (0.12-8.23 mg/g) than anaerobic digestion. The co-fermentation of wheat straw pretreated with 1% H2SO4 resulted in 14.18-46.12% hemicellulose degradation. Co-aerobic straw composting with H2SO4 and K2CO3 at 35 °C reduced the crystallinity of the straw substrate by 6.66 and 7.33%, respectively, as compared to other conditioning agents. CH3COOH lowered the electrical conductivity values of the straw substrate at the end of fermentation (2.33-3.49 mS/cm). Overall, the findings revealed that CH3COOH-cooperative aerobic composting pretreatment at 35 °C is a suitable replacement for the traditional composting process as a method of utilizing straw substrate.

Catalytic mechanism of N-containing biochar on volatile-biochar interaction for the same origin pyrolysis.

Nitrogen, as a common element, is widely present in biomass. The effects of nitrogenous substances on the same origin pyrolysis of biomass and the consequences of N-containing biochar on the catalytic process of volatiles are important for further analyzing the pyrolysis mechanism of biomass. In this research, N-containing biochar was prepared under different conditions, and the interaction between N-containing biochar and biomass pyrolysis volatiles at 400-700 °C was studied. The results show that N-containing biochar can simultaneously participate in reactions as adsorbents, catalysts, and reactants. Its catalytic effect is obviously different for various N configurations. Pyridinic N and pyrrolic N can promote the cracking of lignin into methoxy phenol compounds and promote the further cracking of 5-hydroxymethylfurfural. Graphitic N and oxidized N can promote the further decomposition of phenol and the conversion of D-xylose into small-molecule ketones. In addition, oxidized N can also inhibit the cracking of lignin to produce guaiacol. In the long-term interaction, the highly active pyridinic N tends to convert to a more stable graphitic N.

Preparation and formation mechanism of biomass-based graphite carbon catalyzed by iron nitrate under a low-temperature condition.

Graphite is a widely used industrial material, which experienced a marked shortage caused by the growing demand for electrode anode material and the increased costs for raw material. Graphitic carbon from biomass is a promising approach that will result in low-cost and efficient preparation. Herein, Fe(NO3)3 was selected as the catalyst for pine sawdust, and the effects of temperature and iron content on the graphitization of biochar were investigated. Additionally, the formation mechanism of the graphitic crystallite structure was explored. Results showed that the formation of pyrolysis gas increased with the increase in the amount of catalyst added or pyrolysis temperature. The change in pyrolysis gas, such as H2 and CO, was a critical auxiliary factor reflecting the conversion process. As temperature was increased from 600 °C to 800 °C, the solid products showed high graphitization and low solid yield. Graphite structure mainly formed at 700 °C because of the formation of Fe nanoparticles. The increase in the amount of catalyst could provide more reaction sites and promote the contact between Fe and C, showing that amorphous carbon is dissolved on Fe nanoparticles and precipitated into ordered graphitic carbon. On this basis, a mechanism of "carbon dissolution-precipitation" was proposed to explain the formation of graphite structure, and the whole pyrolysis process included the transformation of the iron element were analyzed.

Effects of three major nutrient contents, compost thickness and treatment time on larval weight, process performance and residue component in black soldier fly larvae (Hermetia illucens) composting.

The cellulose content in vegetable waste (VW) is high and cannot be directly digested by black soldier fly larvae (BSFL). In this study, in order to treat VW using BSFL composting, kitchen waste (KW) is used as the only nutritional supplement for VW to analyze the effects of the different contents of crude protein (CP), crude fat (EE), carbohydrate (3C), compost thickness (CT), and treatment time on the larval weight (LW), survival rate (SR), dry matter reduction rate (DMR), bioconversion rate (BCR), physical and chemical properties of BSFL sand and changes in the microbial community. Our results showed that when the average 3C content increased by 40%, the average LW increased by 47.6%, and the SR, DMR, BCR, and organic matter (OM) content increased by 16.82%, 8.5%, 4.77%, and 3.86%, respectively. In contrast, when the average compost thickness increased by 5 cm, the average weight of BSFL decreased by 22.64%, while the SR of larvae, DMR, BCR, OM, and total nutrients (TN + P2O5 + K2O) decreased by 5%, 5.2%, 4.42%, 9.6%, and 0.78%, respectively. Germination test showed that BSFL sand alone could not be used as soilless culture substrate. After BSFL treatment, we found that the dominant phyla in BSFL sand were Firmicutes (95.77%), Proteobacteria (2.54%), Actinobacteria (0.74%), and Chloroflexi (0.6%). Our results indicate that BSFL composting is an effective method of treating VW, and 3C content and CT have a significant effect on BSFL composting.

Spin crossover dynamics studies on the thermally activated molecular oxygen binding mechanism on a model copper complex.

The theoretical description of the primary dioxygen (O2) binding and activation step in many copper or iron enzymes, suffers from the intrinsically electronic non-adiabaticity of the spin flip events of the triplet dioxygen molecule (3O2), mediated by spin-orbit couplings. In this work, we presented the early-stage ultrafast spin flip dynamics of O2 binding for a simplified monocopper complex, involving the coupled singlet and triplet electronic states. The on-the-fly trajectory surface hopping (TSH) simulations have identified the dynamical effects that may influence the mode of O2 coordination (end-on vs. side-on), and the electronic structures can be viewed as complexes of molecular O2 with Cu(i) or as Cu(ii)-superoxide compounds. In addition, significant spin flip events are obversed within the sub-picosecond regime. We hope this work may provide complimentary insights on the traditional interpretation of O2 binding on copper complexes and subsequent catalytic reaction mechanisms.

Effects of process parameters on the distribution characteristics of inorganic nutrients from hydrothermal carbonization of cattle manure.

In this study, dairy manure was converted into solid and liquid products via hydrothermal carbonization (HTC) to determine optimal reaction conditions to retain the greatest amounts of inorganic nutrients. The influence of three parameters, reaction temperature (150-270 °C), residence time (0.5-6 h) and manure/water ratio (5/200-25/200 g/mL), on the total nutrient content (TNC) in the solid and liquid products were investigated using a 5-level, 3-factor orthogonal test and principal components analysis (PCA). Also, the distribution characteristics of inorganic nutrients were determined using single factor tests. The maximum TNC of the solid product was 22.92 mg/g, obtained at manure/water ratio of 20/200 g/mL and performed at 240 °C for 6 h. For obtaining the maximum TNC of liquid product, the most optimal conditions were 150 °C, 1 h and 20/200 g/mL which produced TNC of 1619.55 mg/L. The proportion of ammonium (NH4+-N) and orthophosphate (PO43-) were also analyzed in the liquid product. The amount of NH4+-N increased with reaction temperature increasing and manure/water ratio decreasing, the main form of phosphorus (P) was PO43- in the liquid product, and most of the potassium (K) were also dissolved in the liquid product. These results indicate that the HTC could be a promising approach for the utilization of dairy manure as inorganic fertilizer in the future.

Addition of plantation waste to the bioconversion of pig manure by black soldier fly larvae: Effects on heavy metal content and bioavailability.

During the conversion of pig manure by black soldier fly larvae (BSFL), the accumulation and speciation changes of heavy metals (HMs) have adverse effects on the environment. In this study, corn straw, rice straw, bamboo chips (BC), wood chips, and rice husk char were added to a bioconversion system to study the accumulation, migration, speciation changes, and microbial correlations of HMs. The results indicated that the addition of BC was most beneficial for the accumulation of HMs (47-72 %) in the BSFL body. In the BC group, the accumulation effect of the BSFL body on zinc (Zn) and arsenic (As) was the most evident (72 and 71 %, respectively). The results of linear fitting (R2 > 0.90) and redundancy analysis (RDA; 90 %) indicated that the bacterium Bacillaceae (Bacillus) was beneficial for increasing the larval weight (LW) of BSFL, and a higher LW accumulated HMs. The addition of BC helped reduce the total amount (6-51 %) of available states (weak acid extraction and reducible states) in the BSFL residue. The RDA results indicated that bacteria (55-92 %) affected the transformation of HM speciation. For example, Zn and cadmium were mainly affected by Firmicutes, whereas copper and chromium were affected by Bacteroidetes. Proteobacteria and Pseudomonas formosensis affected the conversion of lead and As. This study provides important insights into the adsorption of HMs from pig manure by BSFL.

Biotransformation of Pb and As from sewage sludge and food waste by black soldier fly larvae: Migration mechanism of bacterial community and metalloregulatory protein scales.

The accumulation and transformation of lead (Pb) and arsenic (As) during the digestion of sewage sludge (SS) by black soldier fly larvae (BSFL) remain unclear. In this study, we used 16 s rRNA and metagenomic sequencing techniques to investigate the correlation between the microbial community, metalloregulatory proteins (MRPs), and Pb and As migration and transformation. During the 15-day test period, BSFL were able to absorb 34-48 % of Pb and 32-45 % of As into their body. Changes in bacterial community abundance, upregulation of MRPs, and redundancy analysis (RDA) results confirmed that ZntA, EfeO, CadC, ArsR, ArsB, ArsD, and ArsA play major roles in the adsorption and stabilization of Pb and As, which is mainly due to the high contribution rates of Lactobacillus (48-59 %) and Enterococcus (21-23 %). Owing to the redox reaction, the regulation of the MRPs, and the change in pH, the Pb and As in the BSFL residue were mainly the residual fraction (F4). The RDA results showed that Lactobacillus and L.koreensis could significantly (P < 0.01) reduce the reducible fraction (F2) and F4 of Pb, whereas Firmicutes and L.fermentum can significantly (P < 0.05) promote the transformation of As to F4, thus realizing the passivation Pb and As. This study contributes to the understanding of Pb and As in SS adsorbed by BSFL and provides important insights into the factors that arise during the BSFL-mediated migration of Pb and As.

Optimizing biochar addition strategies in combined processes: Comprehensive assessment of earthworm growth, lignocellulose degradation and vermicompost quality.

The sustainable management of agricultural waste is essential for curtailing environmental contamination. To address the shortcomings of single treatment methods, this study evaluated the feasibility of combining membrane-covered composting (MC) with vermicomposting. Based on this, the integrated effects of different biochar addition strategies on the combined process were investigated. The aim was to improve the efficiency of vermicomposting while eliminating the negative effects of biochar on earthworms. Addition of biochar before membrane-covered composting increased total earthworm biomass by 25.6 - 31.4 % and reproduction rate by 13.4 - 23.9 %. Specifically, the electrical conductivity (EC) (1061.0 - 1112.0 uS/cm) of the vermicompost was significantly reduced, while the total nutrient content (42.3 - 42.6 mg/g) and germination index (GI) (103.9 - 108.4 %) were maximized. Additionally, reductions in the carbon-to-nitrogen ratio and volatile content were observed. Overall, combination process is a promising approach to improve the quality of vermicomposting. The study's results offer a novel perspective on the value-added treatment of agricultural waste.

Performance evaluation of a solar evaporation system for liquid digestate concentration.

The safe and efficient treatment and resource recovery of the large amount of liquid digestate (LD) produced by biogas plants has become an important factor limiting the development of the biogas industry. In this study, a solar evaporation system was developed based on low cost, high efficiency, and high nutrient recovery of LD concentration. The effects of different parameters, including solar radiation, LD flowrate, and daily treatment capacity, on the concentration efficiency, nutrient recovery, and thermal efficiency were investigated. Economic and enviroeconomic analyses were conducted to comprehensively assess the feasibility and environmental benefits of the system for large-scale biogas plants. The results showed that the higher the solar radiation, the higher the air temperature and its saturation vapor pressure, which led to an increase in the concentration efficiency of LD. The LD concentration efficiency can reach up to 43.33% when the average daily solar radiation was 773.08 W/m2. High solar radiation also contributed to high LD temperature, leading to an increase in the percentage of free ammonia (NH3) in the LD, which was not conducive to the recovery of ammonia nitrogen (NH3N). By increasing the LD flow rate, the heat and mass exchange between air and LD in the spray chamber was enhanced, resulting in higher concentration efficiency. The variation of LD flow rate and daily treatment capacity had little effect on nutrient recovery. The recovery of NH3N and soluble salts in the concentrated phase could be as high as 78.39% and 84.19%, respectively, when the initial pH of LD was 5, the air flow rate was 0.03 m3/s, and the LD flow rate was 6 L/min. In addition, the economic analysis indicated that the concentration cost of the system in treating a daily LD production of 100 tons was 1.08 USD/ton. The solar evaporation system can reduce CO2 emissions by 2.40 × 105 tons and earn carbon credits of 3.48 × 106 USD over its lifetime. This study provides a new direction and reference for LD concentration.

Biorefining waste into nanobiotechnologies can revolutionize sustainable agriculture.

Modern agriculture has evolved technological innovations to sustain crop productivity. Recent advances in biorefinery technology use crop residue as feedstock, but this raises carbon sequestration concerns as biorefining utilizes carbon that would otherwise be returned to the soil, thus causing a decline in crop productivity. Furthermore, biorefining generates abundant lignin waste that significantly impedes the efficiency of biorefineries. Valorizing lignin into advanced nanobiotechnologies for agriculture provides a unique opportunity to balance bioeconomy and soil carbon sequestration. Integration of agricultural practices such as utilization of agrochemicals, fertilizers, soil modifiers, and mulching with lignin nanobiotechnologies promotes crop productivity and also enables advanced manufacturing of high-value bioproducts from lignin. Lignin nanobiotechnologies thus represent state-of-the-art innovations to transform both the bioeconomy and sustainable agriculture.

Enrichment and speciation changes of Cu and Cd in black soldier fly (Hermetia illucens) larval compost and their effects on larval growth performance.

Municipal sludge (MS), rainwater sludge (RS), and kitchen waste (KW) were used as nutritional supplements for black soldier fly larvae (BSFL). Cd (52.3 %) was more easily assimilated in the BSFL body than Cu (34.8 %). After biotransformation in BSFL, the weak acid-soluble fraction (F1) of Cu and Cd increased by an average of 29.0 % and 42.7 %, respectively, whereas the reducible fraction (F2) of Cu and Cd decreased by an average of 13.8 % and 56.4 %, respectively, in the BSFL sand (BSFL feces and waste residues). A significant correlation (P < 0.01) was found between pH and the speciation of Cu and Cd. The abundance of Bacteroides had a positive correlation (P < 0.05) with the F1 of Cu, an extremely significant negative correlation (P < 0.001) with the F2 of Cd, and an extremely significant positive correlation with the F1 of Cd (P < 0.001). In addition, Cu and Cd exposures significantly (P < 0.01) reduced larval weight by 67.7 % and 45.3 %, respectively, pupation rate by 46.3 % and 26.5 %, respectively, and eclosion rate by 35.5 % and 33.4 %, respectively. Exposure to high concentrations of Cu and Cd also prolonged the development cycle (1-12 days) of BSFL and led to the failure of BSFL to complete their metamorphosis.

Effects of segmented aerobic and anaerobic fermentation assisted with chemical treatment on comprehensive properties and composition of wheat straw.

Traditional aerobic composting used for straw treatment shows limited regulation effects and unstable properties, and it is necessary to introduce some co-processing methods to optimize its performance. Herein, segmented aerobic/anaerobic fermentation, combined with chemical treatment with wood vinegar/NaOH, was used to treat wheat straw. The results showed that anaerobic fermentation when used as the first stage could stabilize the wheat straw pH between 5.19 and 6.13 and improve nutrient contents. All treatments had greater effects on substrate aeration porosities (range of 14%) than on total porosity (range of 6%), and the water-holding porosities were improved to a greater extent by NaOH than by wood vinegar. The hemicellulose degradation rate of aerobic-anaerobic treatment was higher than that achieved with anaerobic-aerobic treatment, while the latter method was more effective at removing the neutral detergent-soluble as well as remaining organic matter, which was generated due to a higher KCl content in the ash.

A novel technology for separating live earthworm from vermicompost: Experiment, mechanism analysis, and simulation.

The vermicomposting is widely acknowledged as an effective and sustainable biological technology to dispose large scale organic solid waste such as livestock manure, crop residues and municipal sludge. The scale of vermicomposting has constantly increased over recent years due to high market demand for live earthworms. Rapid and efficient extraction and harvesting earthworm from large-scale vermicompost has become a bottle-neck problem at the end of vermicomposting. Currently, most earthworm separation is done manually or relies on simple tools, and is therefore low efficient, time consuming and labor intensive. In this study, to explore earthworm separation technology and underlying mechanisms, three major studies were conducted based on the developed separator: Earthworm separation experiment, mechanism analysis of separation, and separation process simulation. The result indicated that under a 45% moisture content of vermicompost, earthworm can be separated centrifugally with approximately 83.79% worm separation rate. The treatment capacity of separator is 21.2 kg of total vermicompost mixture per minute. The critical factor in earthworm-vermicompost separation is frictional force and liquid membrane adhesion at the contact interface of conical separator. The final separated X velocity of earthworms is higher, whereas the Y and Z velocities are less than those of vermicompost. The earthworms are centrifugally thrown to a specific area called the earthworm harvest position. The outcome of this study provides a valuable reference for improvement of earthworm harvest efficiency and for shortening the recycling period of vermicomposting products in practice.

Effects of different fermentation assisted enzyme treatments on the composition, microstructure and physicochemical properties of wheat straw used as a substitute for peat in nursery substrates.

To solve the central problems caused by traditional composting treatments, such as long-time consumption and poor regulation effects, this study used three fermentation methods and four enzymes to develop rapid and directional regulation methods to convert wheat straw into a suitable substrate. The results showed that the mixed anaerobic method led to better pH (4.39-5.75) and EC values (1.27-1.89 mS/cm) in the straw substrates, while the aerobic method retained more nutrients and increased lignin and cellulose contents by 5.07-8.04% and 1.52-3.32%. The cellulase mixed with hemicellulase or laccase treatments all increased the crystallinity by 0.45-7.23%. The TG/DTG results showed that all treatments decreased the initial straw glass transition temperature, particularly when using the mixed anaerobic method, with decreases of 10.63-25.48 °C. Overall, mixed anaerobic fermentation and multiple enzymes, including cellulase, have been suggested as alternative biological modification methods for straw substrates.

Influence of the structure and properties of lignocellulose on the physicochemical characteristics of lignocellulose-based residues used as an environmentally friendly substrate.

The extensive use of nonrenewable peat does not meet the strategic goals of sustainable development. This study explores the advantages and disadvantages of using lignocellulose-based agricultural and forestry wastes as peat substitute in substrates for soilless cultivation; further, it also investigates the key factors influencing the physical and chemical properties of the substrates. Accordingly, the physical and chemical properties of four gramineous crop straws and two woody forestry wastes were determined and compared with those of peat and coconut bran. In addition, cellulose, hemicellulose, and lignin were extracted from wheat straw and pine sawdust, and their basic characteristics and structures were compared and analyzed. The results showed that the influence of particle size on the physical properties of substrates was significantly higher (P < 0.01) than the influence of the substrate type, especially with respect to the water-holding and aeration porosities, which had effect sizes (Eta2) of 73.8% and 68.2%, respectively. The electrical conductivity values of the four straws (1.87-3.42 mS/cm) were higher than those of peat and coconut bran (0.50-0.96 mS/cm), which was mainly due to the high hemicellulose contents (28.52%-30.10%) and total nutrient contents (28.46-47.81 g/kg) of the straws. In contrast, the electrical conductivity values of the woody waste substrates were lower (0.28-0.33 mS/cm) than those of peat and coconut bran. Peat and coconut bran contained the lowest cellulose (17.84%-20.95%) and hemicellulose contents (5.14%-7.19%) of all substrates, resulting in a low degradability and good stability. The crystallinity of coconut bran (23.06%) was significantly lower than that of all other substrates (30.36%-43.03%), which mainly contributed to the superior compressibility of coconut bran. The best pretreatment method for biomass waste used as a substrate should be selected according to the target properties of the corresponding components.

Investigation of physico-chemical properties of hydrochar and composition of bio-oil from the hydrothermal treatment of dairy manure: Effect of type and usage volume of extractant.

Hydrothermal treatment for dairy manure into value-added hydrochar and bio-oil is a potential technology for its resource utilization. During the process of treatment, extractant is applied to the separation of hydrochar and bio-oil. In this study, three polar extractants (ethyl acetate, dichloromethane, diethyl ether) and two nonpolar extractants (n-hexane and petroleum ether) were used, and the physico-chemical properties of hydrochar and the composition of bio-oil were investigated. Compared with nonpolar extractants, polar extractants could extract the bio-oil absorbed on the hydrochar exterior and interior surface, resulting in more mass loss of hydrochar and better extraction performance on the production of bio-oil. The decrease of H/C atomic ratio and the increase of O/C atomic ratio indicated the demethanation tendency to occur during the extraction process, and enhanced the hydrochar stability. The scanning electron microscope and specific surface area analysis revealed that polar extractant had a more positive effect than nonpolar extractant on the occurrence of disperse spherical microparticles and the augment of hydrochar specific surface area. The bio-oil from polar extractant mainly consisted of N-containing compounds, ketones, phenols and acids, while the bio-oil from nonpolar extractant mainly consisted of esters, alkanes and aromatics. These results reveal that the hydrochar extracted by polar solvent exerts a greater potential for the production of carbon-based material.

Enhancement of biogas production from rape straw using different co-pretreatment techniques and anaerobic co-digestion with cattle manure.

The present study investigated the possibility of valorizing rape straw through anaerobic digestion and the possibility of improving biomethane yield by pretreatment with H2SO4, combined H2SO4 with steam explosion (SE) and SE combined with superfine grinding (SFG). To evaluate the pretreatment method efficiency, several analytical techniques were applied. Additionally, the performance of co-digesting of cattle manure (CM) with pretreated rape straw (PRS) at different ratios was evaluated. The results showed that combined pretreatment could dissolve the lignocellulosic fiber structure, which positively stimulated methane yield. The highest cumulative CH4 yield (CMY) of 305.7 mLg-1VS was achieved by combined SE at 180 °C for 5 min with SFG, which was 77.84% higher than the untreated. The CMY was further improved by 11.4-59% higher than the control (CM) using co-digestion. This study confirmed that, under optimal parameters of AD, pretreatment with SEG180 could significantly boost the CMY from co-digestion of CM and PRS.

How a single 5-methylation of cytosine regulates the recognition of C/EBPß transcription factor: a molecular dynamic simulation study.

CpG methylation can regulate gene expression by altering the specific binding of protein and DNA. In order to understand how a single 5mC regulates protein-DNA interactions, we have compared the structures and dynamics of CEBP/ßprotein-DNA complexes before and after methylation, and the results indicate that even a single 5mC can regulate protein-DNA recognition by steric-hindrance effect of methyl group and changing the hydrogen bond interactions. The interactions between the methyl group, mCpG motif, and the conserved residue arginine make the protein read out the variation of local environment, which further enhances the specific recognition and affects the base pair stacking. The stacking interactions can propagate along the backbone of DNA and lead to long-range allosteric effects, including obvious conformational variations for DNA base pairs.