Liquid fertilizers produced by microwave-assisted acid hydrolysis of livestock and poultry wastes and their effects on hot pepper cultivation.

Liquid fertilizers (LFs) produced by microwave-assisted acid hydrolysis of livestock and poultry wastes were applied to potted hot pepper (Capsicum annuum L.) to evaluate their potential to be used as amino acid LFs. A preliminary experiment was conducted to determine the optimum acid-hydrolysis conditions for producing LFs from a mixture of pig hair and faeces (P) and another mixture of chicken feathers and faeces (C). Two LFs were produced under the optimum acid-hydrolysis conditions (acidification by sulphuric acid (7.5 mol L-1) in a microwave (200 W) for 90 minutes), and a commercial amino acid LF (Guo Guang (GG)) was used for comparison. P, C and GG fertilizers were tested in potted hot pepper cultivation at two doses, whereas no fertilizer application served as the control (CK). P and C fertilizers significantly increased the fruit yield compared with GG fertilizer, particularly at the higher dose. Moreover, the treatments improved the fruit vitamin C and soluble sugar contents in the order of C > P > GG compared with CK. These results could be attributed to more types of amino acids in C fertilizer than in P and GG fertilizers. The results also indicated that the prepared fertilizers could significantly increase the shoot and root dry weight, soil available nitrogen and phosphorus contents and nitrogen, phosphorus, and potassium (NPK) uptake by plants compared with CK. In conclusion, microwave-assisted acid hydrolysis could effectively convert unusable wastes into valuable fertilizers comparable or even superior to commercial fertilizers.

Intervention effects of fructooligosaccharide and astragalus polysaccharide, as typical antibiotic alternatives, on antibiotic resistance genes in feces of layer breeding: advantages and defects.

Although antibiotic alternatives are widely used in livestock and poultry breeding industry after in-feed antibiotics ban, their intervention effects on antibiotic resistance genes (ARGs) in these food animals' feces remain poorly understood. Here effects of fructooligosaccharide (FOS) and astragalus polysaccharide (APS), as typical antibiotic alternatives in China, on ARGs in layer feces were estimated by performing metagenomic sequencings and fluorescence quantitative PCR. Fructooligosaccharide significantly reduced sum abundance of ARGs and mobile genetic elements (MGEs) by increasing Lactobacillus clones and reducing Escherichia clones which had relatively higher abundances of ARG subtypes and MGE subtypes in layer feces. However, at least parts of core ARGs and MGEs categories were not reduced by FOS, such as aminoglycosides- and tetracyclines-resistant genes, Tn916, Integrase, and so on. MGEs and microbiome, especially Escherichia genus and Lactobacillus genus, were the key factors affecting ARGs' sum abundance. MGEs had a higher correlation coefficient with ARGs' sum abundance than Escherichia genus and Lactobacillus genus. These findings firstly reveal the defects of antibiotic alternatives in controlling bacterial resistance in livestock and poultry breeding after in-feed antibiotics ban, and more strategies are needed to control pollutions and risks of core ARGs and MGEs in food animals' feces under a special environment.

Crop effect and mechanism of amino acid-modified biomass ash in remediation of cadmium-contaminated soil.

Lignocellulosic biomass ash (BA) has certain adsorption and passivation effects on heavy metals, but its function is generally weak. Amino acid salt can facilitate the leaching of heavy metals in soil. Therefore, modification of BA with amino acid salt may realize a higher leaching rate and better passivation of heavy metals in soil. In this study, BA was modified by amino acid hydrolysate obtained from the hydrolysis of chicken feathers by sulfuric acid. The physicochemical properties of BA and modified BA (MBA), their effects on Chinese cabbage (CC) yield and nutritional quality, and passivation effects on soil cadmium (Cd) were compared, and the related mechanisms were investigated. SEM-EDS, XRD, and FTIR demonstrated that BA was a CaCO3-type soil amendment, while MBA was a CaSO4-type soil amendment with the loading of amino acid. Compared with BA, MBA significantly increased the fresh weight, soluble sugar, vitamin C (Vc), and protein contents of CC in both non-Cd contaminated soil and Cd contaminated soil, and obviously decreased the nitrate content and Cd uptake of CC in Cd-contaminated soil. After the application of MBA, cadmium species in potted soil were transformed from higher plant availability, representing by exchangeable and carbonate-bound, into lower plant availability, representing by iron-manganese oxide bound, which was identified as the key reason for the significant reduction of Cd content in CC under MBA application.

Efficiency and mechanism in preparation and heavy metal cation/anion adsorption of amphoteric adsorbents modified from various plant straws.

Cellulose can be modified for the loading of functional groups such as amino groups, sulfydryl groups, and carboxyl groups. Cellulose-modified adsorbents generally have specific adsorption capacities for either heavy metal anions or cations, and possess the advantages of wide raw material source, high modification efficiency, high adsorbent recyclability, and great convenience in recovery of the adsorbed heavy metals. At present, preparation of amphoteric heavy metal adsorbents from lignocellulose has attracted great attention. However, the difference in efficiency of preparing heavy metal adsorbents by modification of various plant straw materials and mechanism for the difference remain to be further explored. In this study, three plant straws, including Eichhornia crassipes (EC), sugarcane bagasse (SB) and metasequoia sawdust (MS), were sequentially modified by tetraethylene-pentamine (TEPA) and biscarboxymethyl trithiocarbonate (BCTTC) to obtain amphoteric cellulosic adsorbents (EC-TB, SB-TB and MS-TB, respectively), which can simultaneously adsorb heavy metal cations or anions. The heavy metal adsorption properties and mechanism before and after modification were compared. Pb(II) and Cr(VI) removal rates by the three adsorbents were 2.2-4.3 folds and 3.0-13.0 folds of those before modification, respectively, following the order of MS-TB > EC-TB > SB-TB. In the five-cycle adsorption-regeneration test, the Pb(II) and Cr(VI) removal rate by MS-TB decreased by 58.1 % and 21.5 %, respectively. Among the three plant straws, MS possessed the most abundant hydroxyl groups and the largest specific surface area (SSA), and accordingly MS-TB had the highest load of adsorption functional groups [(C)NH, (S)CS and (HO)CO] and also the largest SSA among the three adsorbents, which contribute to its highest modification and adsorption efficiency. This study is of great significance for screening suitable raw plant materials to prepare amphoteric heavy metal adsorbents with superior adsorption performance.

Effects of polylactic acid (PLA) and polybutylene adipate-co-terephthalate (PBAT) biodegradable microplastics on the abundance and diversity of denitrifying and anammox bacteria in freshwater sediment.

Microplastics (MPs) have been widely distributed on Earth and have drawn global concern for freshwater and marine ecosystems. Biodegradable plastics have risen in popularity to replace nonbiodegradable plastics all over the world. The effects of biodegradable plastics on denitrifying and anammox bacteria in freshwater sediment remain largely unknown. In this study, water column reactors containing polylactic acid (PLA) or polybutylene adipate-co-terephthalate (PBAT) MPs in sediment were established to simulate lake ecosystems and analyze the effects of biodegradable MPs on sedimentary nitrogen transformation microorganisms. The total organic carbon (TOC) concentrations in the PLA and PBAT groups were slightly higher than those in the control group, which might be related to the slow degradation of these two plastics. Denitrifying and anammox bacterial diversities decreased after adding MPs to sediments for 30 days, and the dominant OTUs of these two bacteria were differentiated from the control. The abundance levels of nirS denitrifying and anammox bacteria on the PLA MP surface were significantly higher than those in the other groups (P < 0.05), but they were lower in the PBAT groups than in the other groups. As an excellent electron donor for the denitrification process, lactic acid release from PLA degradation resulted in the enrichment of denitrifying and anammox bacteria on the MP surfaces. However, PBAT led to various responses of bacteria in an anaerobic environment. In addition, the redundancy analysis results indicated that total phosphorus, TOC and nitrate were strongly negatively correlated with the abundance levels of denitrifying and anammox bacteria. Our findings provided insight into the effects of MPs, especially the biodegradable ones, on sedimentary nitrogen-transformation bacteria.

SSThe coexistence and diversity of Candidatus methylomirabilis oxyfera-like and anammox bacteria in sediments of an urban eutrophic lake / SLa coexistencia y diversidad de bacterias Candidatus methylomirabilis oxyfera y anammox en sedimentos de un lago urbano eutrófico

Tangxun Lake is the largest urban lake in China, which is polluted by multiple wastewaters, and now is severely eutrophic. We detected diversity, abundance, and the coexistence of Candidatus Methylomirabilis oxyfera-like and anammox bacteria in different horizontal and vertical directions of the lake sediments through qPCR and clone library. Phylogenetic tree analysis showed that the Ca. Methylomirabilis oxyfera-like and anammox bacteria exhibited high diversity, and they belonged to group B—E and Ca. Brocadia genus, respectively. These two bacteria displayed higher diversity in polluted area than in other areas. Furthermore, they had great spatial variation of abundance both horizontally and vertically. The abundance of anammox bacteria was significantly higher than that of Ca. Methylomirabilis oxyfera-like bacteria. The stronger the human interference were, the higher abundances these two bacteria exhibited horizontally, whereas both their abundances and the ratio of anammox to Ca. Methylomirabilis oxyfera-like bacteria decreased with the increasing depth. Redundancy analysis indicated that nitrate was the most influential environmental factor to the abundance of these two bacteria. Ammonia, nitrite, total nitrogen, and organic matters were in positive correlation with the abundance of these two bacteria. Nitrate was slightly negatively correlated with the abundance of Ca. Methylomirabilis oxyfera-like bacteria, while it was positively correlated with that of anammox bacteria. Our results provided an insight into the effects of environmental factors such as ammonia, nitrite, and nitrate on the diversity and abundances of these two bacteria and theoretical basis for restoration of water.(AU)

SSThe coexistence and diversity of Candidatus methylomirabilis oxyfera-like and anammox bacteria in sediments of an urban eutrophic lake.

Tangxun Lake is the largest urban lake in China, which is polluted by multiple wastewaters, and now is severely eutrophic. We detected diversity, abundance, and the coexistence of Candidatus Methylomirabilis oxyfera-like and anammox bacteria in different horizontal and vertical directions of the lake sediments through qPCR and clone library. Phylogenetic tree analysis showed that the Ca. Methylomirabilis oxyfera-like and anammox bacteria exhibited high diversity, and they belonged to group B-E and Ca. Brocadia genus, respectively. These two bacteria displayed higher diversity in polluted area than in other areas. Furthermore, they had great spatial variation of abundance both horizontally and vertically. The abundance of anammox bacteria was significantly higher than that of Ca. Methylomirabilis oxyfera-like bacteria. The stronger the human interference were, the higher abundances these two bacteria exhibited horizontally, whereas both their abundances and the ratio of anammox to Ca. Methylomirabilis oxyfera-like bacteria decreased with the increasing depth. Redundancy analysis indicated that nitrate was the most influential environmental factor to the abundance of these two bacteria. Ammonia, nitrite, total nitrogen, and organic matters were in positive correlation with the abundance of these two bacteria. Nitrate was slightly negatively correlated with the abundance of Ca. Methylomirabilis oxyfera-like bacteria, while it was positively correlated with that of anammox bacteria. Our results provided an insight into the effects of environmental factors such as ammonia, nitrite, and nitrate on the diversity and abundances of these two bacteria and theoretical basis for restoration of water.

Effects of illumination on nirS denitrifying and anammox bacteria in the rhizosphere of submerged macrophytes.

Visibility in lakes can decrease due to increases in the amounts of suspended solids and algae, which inhibits the growth of submerged macrophytes. However, the understanding about whether illumination reduction affects the nitrogen-cycling microorganisms in the rhizosphere of submerged macrophytes, is limited. The abundance and biodiversity of nirS denitrifying and anammox bacteria in the rhizosphere of Potamogeton crispus were studied under 0% (natural light), 20%, 40%, and 60% shading treatments. The abundance of the nirS gene was highest under 60% shading treatment, while the anammox 16S rRNA gene was highest under 40% shading treatment. Moreover, the abundance of the two genes were lower under natural light than under shading conditions during most sampling periods. The quantitative ratio of the two gene (anammox 16S rRNA to nirS gene) abundance fluctuated wildly with the distance away from the roots, under natural light and 20% shading treatment. However, the ratio varied relatively little under 40% and 60% shading treatments. The diversity of nirS denitrifying bacteria was high in the rhizosphere, while the diversity of anammox bacteria was low, and Candidatus Brocadia fulgida was dominant. This study revealed that illumination reduction not only facilitated the growth of nirS denitrifying and anammox bacteria in the rhizosphere, but also weakened the competition between the two bacteria.

Comparison of Fenton and bismuth ferrite Fenton-like pretreatments of sugarcane bagasse to enhance enzymatic saccharification.

This study compared enzymatic saccharification of sugarcane bagasse (SCB) after application of two different pretreatment methods, Fenton pretreatment (FP) and BiFeO3 Fenton-like pretreatment (BFP). The composition, morphology and structural properties of SCB with different pretreatments were analyzed. Results showed that, after BFP, the yield of reducing sugar of SCB under enzymatic saccharification for 72 h was 25.8%, and the sugar conversion rate was 36.6%, which were 2.2 and 2.4-fold those of the FP, respectively. Moreover, the removal of hemicellulose and delignification in the BFP was more severe than that in the FP. The determination of hydroxyl radical (OH) in the two different Fenton processes revealed that the OH generated in the BiFeO3 Fenton-like system was higher in concentration and longer in action time than that in the Fenton system, which was likely key to the stronger effect of BFP than FP on the enzymatic saccharification of SCB.

Nitrifying aerobic granular sludge fermentation for releases of carbon source and phosphorus: The role of fermentation pH.

The effect of fermentation pH (uncontrolled, 4 and 10) on the releases of carbon source and phosphorus from nitrifying aerobic granular sludge (N-AGS) was investigated. Meanwhile, metal ion concentration and microbial community characterization were explored during N-AGS fermentation. The results indicated that N-AGS fermentation at pH 10 significantly promoted the releases of soluble chemical oxygen demand (SCOD) and total volatile fatty acids (TVFAs). However, SCOD and TVFA released from N-AGS were inhibited at pH 4. Moreover, acidic condition promoted phosphorus release (mainly apatite) from N-AGS during anaerobic fermentation. Nevertheless, alkaline condition failed to increase phosphorus concentration due to the formation of chemical-phosphate precipitates. Compared with the previously reported flocculent sludge fermentation, N-AGS fermentation released more SCOD and TVFAs, possibly due to the greater extracellular polymeric substances content and some hydrolytic-acidogenic bacteria in N-AGS. Therefore, N-AGS alkaline fermentation facilitated the carbon source recovery, while N-AGS acidic fermentation benefited the phosphorus recovery.

Treatment for domestic wastewater from university dorms using a hybrid constructed wetland at pilot scale.

A hybrid constructed wetland (CW) system was operated at pilot scale to evaluate the treatment of domestic wastewater from university dorms in Hubei province, China. The hybrid system consisted of an integrated vertical flow constructed wetland (IVF CW) (a down-flow VF CW and an up-flow VF CW) and a horizontal subsurface flow constructed wetland (HSF CW). The influent for the hybrid CW containing chemical oxygen demand (COD), total phosphorus (TP), total nitrogen (TN), and ammonium nitrogen (NH4+-N) with mean concentrations of 152, 0.75, 18.9, and 8.9 mg L-1, respectively, was purified by the hybrid CW system to achieve mean removal efficiencies of 59.0% COD, 82.8% TP, 57.7% TN, and 79.2% NH4+-N over a 9-month period. The nitrification intensity of the media in the down-flow VF CW was the highest compared to the other CWs, whereas the denitrification intensity of the media in the HSF CW was significantly higher than the IVF CW. The abundance of nitrifying bacteria in the media of the IVF CW was higher than in the HSF CW, while a higher abundance of denitrifying bacteria was found in the HSF CW compared to the IVF CW, suggesting that the HSF CW was more suitable for the growth of denitrifying bacteria.

Analysis of utilization technologies for Eichhornia crassipes biomass harvested after restoration of wastewater.

Eichhornia crassipes (EC, water hyacinth) has gained attention due to its alarming reproductive capacity, which subsequently leads to serious ecological damage of water in many eutrophic lakes in the world. The traditional mechanical removal methods have disadvantages. They squander this valuable lignocellulosic resource. Meanwhile, there is a bottleneck for the subsequently reasonable and efficient utilization of EC biomass on a large scale after phytoremediation of polluted water using EC. As a result, the exploration of effective EC utilization technologies has become a popular research field. After years of exploration and amelioration, there have been significant breakthroughs in this research area, including the synthesis of excellent EC cellulose-derived materials, innovative bioenergy production, etc. This review organizes the research of the utilization of the EC biomass among several important fields and then analyses the advantages and disadvantages for each pathway. Finally, comprehensive EC utilization technologies are proposed as a reference.

Effect of humic acids with different characteristics on fermentative short-chain fatty acids production from waste activated sludge.

Recently, the use of waste activated sludge to bioproduce short-chain fatty acids (SCFA) has attracted much attention as the sludge-derived SCFA can be used as a preferred carbon source to drive biological nutrient removal or biopolymer (polyhydroxyalkanoates) synthesis. Although large number of humic acid (HA) has been reported in sludge, the influence of HA on SCFA production has never been documented. This study investigated the effects on sludge-derived SCFA production of two commercially available humic acids (referred to as SHHA and SAHA purchased respectively from Shanghai Reagent Company and Sigma-Aldrich) that differ in chemical structure, hydrophobicity, surfactant properties, and degree of aromaticity. It was found that SHHA remarkably enhanced SCFA production (1.7-3.5 folds), while SAHA had no obvious effect. Mechanisms study revealed that all four steps (solubilization, hydrolysis, acidification, and methanogenesis) involved in sludge fermentation were unaffected by SAHA. However, SHHA remarkably improved the solubilization of sludge protein and carbohydrate and the activity of hydrolysis enzymes (protease and α-glucosidase) owing to its greater hydrophobicity and protection of enzyme activity. SHHA also enhanced the acidification step by accelerating the bioreactions of glyceradehyde-3P → d-glycerate 1,3-diphosphate, and pyruvate → acetyl-CoA due to its abundant quinone groups which served as electron acceptor. Further investigation showed that SHHA negatively influenced the activity of acetoclastic methanogens for its competition for electrons and inhibition on the reaction of acetyl-CoA → 5-methyl-THMPT, which caused less SCFA being consumed. All these observations were in correspondence with SHHA significantly enhancing the production of sludge derived SCFA.

Stimulating short-chain fatty acids production from waste activated sludge by nano zero-valent iron.

An efficient and green strategy, i.e. adding nano zero-valent iron into anaerobic fermentation systems to remarkably stimulate the accumulation of short-chain fatty acids from waste activated sludge via accelerating the solubilization and hydrolysis processes has been developed. In the presence of nano zero-valent iron, not only the short-chain fatty acids production was significantly improved, but also the fermentation time for maximal short-chain fatty acids was shortened compared with those in the absence of nano zero-valent iron. Mechanism investigations showed that the solubilization of sludge, hydrolysis of solubilized substances and acidification of hydrolyzed products were all enhanced by addition of nano zero-valent iron. Also, the general microbial activity of anaerobes and relative activities of key enzymes with hydrolysis and acidification of organic matters were improved than those in the control. 454 high-throughput pyrosequencing analysis suggested that the abundance of bacteria responsible for waste activated sludge hydrolysis and short-chain fatty acids production was greatly enhanced due to nano zero-valent iron addition.

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type.

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

Altering protein conformation to improve fermentative hydrogen production from protein wastewater.

One important reason for low hydrogen production from protein wastewater is due to the native folded conformation of protein. In this study the enhancement of bio-hydrogen production from protein wastewater by altering protein conformation via pretreatment was reported. Firstly, the effect of different pretreatment methods (acid, alkali, heat, and ultraviolet) on hydrogen production from synthetic protein wastewater was compared. The hydrogen production from the ultraviolet pretreated wastewater was 111.3 mL/g-protein, which was 3.79-, 3.73-, 3.54-, and 1.36-fold of that from the unpretreated (blank), acid, alkali, and heat pretreated wastewater, respectively. Then, the reasons for ultraviolet pretreatment showing significantly higher hydrogen production than other pretreatments were investigated. It was found that all pretreatments did not cause the cleavage of peptide bond, but the ultraviolet one caused much greater damage of hydrogen bonding networks and unfolding of protein. Thus, during anaerobic fermentation much higher protease activity and protein utilization were observed, which resulted in the bio-hydrogen production being remarkably improved. Further studies indicated that the photo-oxidization of aromatic residues in protein was not the reason for ultraviolet pretreatment remarkably improving bio-hydrogen production. Finally, the application of ultraviolet pretreatment to enhance hydrogen production from real protein wastewater was testified.

Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production.

Short-chain fatty acids (SCFAs) have been regarded as the excellent carbon source of wastewater biological nutrient removal, and sludge alkaline (pH 10) fermentation has been reported to achieve highly efficient SCFAs production. In this study, the underlying mechanisms for the improved SCFAs production at pH 10 were investigated by using 454 pyrosequencing and fluorescent in situ hybridization (FISH) to analyze the microbial community structures in sludge fermentation reactors. It was found that sludge fermentation at pH 10 increased the abundances of Pseudomonas sp. and Alcaligenes sp., which were able to excrete extracellular proteases and depolymerases, and thus enhanced the hydrolysis of insoluble sludge protein and polyhydroxyalkanoates (PHA). Meanwhile, the abundance of acid-producing bacteria (such as Clostridium sp.) in the reactor of pH 10 was also higher than that of uncontrolled pH, which benefited the acidification of soluble organic substrates. Further study indicated that sludge fermentation at pH 10 significantly decreased the number of methanogenic archaea, resulting in lower SCFAs consumption and lower methane production. Therefore, anaerobic sludge fermentation under alkaline conditions increased the abundances of bacteria involved in sludge hydrolysis and acidification, and decreased the abundance of methanogenic archaea, which favored the competition of bacteria over methanogens and resulted in the efficient production of SCFAs.

Enhancement of hydrogen production during waste activated sludge anaerobic fermentation by carbohydrate substrate addition and pH control.

The effects of carbohydrate/protein ratio (CH/Pr) and pH on hydrogen production from waste activated sludge (WAS) were investigated. Firstly, the optimal pH value for hydrogen production was influenced by the CH/Pr ratio, which was pH 10, 9, 8, 8, 8 and 6 at the CH/Pr ratio (COD based) of 0.2 (sole sludge), 1, 2.4, 3.8, 5 and 6.6, respectively. The maximal hydrogen production (100.6 mL/g-COD) was achieved at CH/Pr of 5 and pH 8, which was due to the synergistic effect of carbohydrate addition on hydrogen production, the enhancement of sludge protein degradation and protease and amylase activities, and the suitable fermentation pathway for hydrogen production. As hydrogen consumption was observed at pH 8, in order to further increase hydrogen production a two-step pH control strategy (pH 8+pH 10) was developed and the hydrogen production was further improved by 17.6%.

Effects of metal oxide nanoparticles (TiO2, Al2O3, SiO2 and ZnO) on waste activated sludge anaerobic digestion.

The effect of metal oxide nanoparticles (nano-TiO2, nano-Al2O3, nano-SiO2 and nano-ZnO) on anaerobic digestion was investigated by fermentation experiments using waste activated sludge as the substrates. Nano-TiO2, nano-Al2O3 and nano-SiO2 in doses up to 150 milligram per gram total suspended solids (mg/g-TSS) showed no inhibitory effect, whereas nano-ZnO showed inhibitory effect with its dosages increased. The methane generation was the same as that in the control when in the presence of 6 mg/g-TSS of nano-ZnO, however, which decreased respectively to 77.2% and 18.9% of the control at 30 and 150 mg/g-TSS. The released Zn2+ from nano-ZnO was an important reason for its inhibitory effect on methane generation. It was found that higher dosages of nano-ZnO inhibited the steps of sludge hydrolysis, acidification and methanation. Also, the activities of protease, acetate kinase (AK) and coenzyme F420 were inhibited by higher dosages of nano-ZnO during WAS anaerobic digestion.