Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Efficient production of l-glutathione by whole-cell catalysis with ATP regeneration from adenosine.

l-glutathione (GSH) is an important tripeptide compound with extensive applications in medicine, food additives, and cosmetics industries. In this work, an innovative whole-cell catalytic strategy was developed to enhance GSH production by combining metabolic engineering of GSH biosynthetic pathways with an adenosine-based adenosine triphosphate (ATP) regeneration system in Escherichia coli. Concretely, to enhance GSH production in E. coli, several genes associated with GSH and  l-cysteine degradation, as well as the branched metabolic flow, were deleted. Additionally, the GSH bifunctional synthase (GshFSA) and GSH ATP-binding cassette exporter (CydDC) were overexpressed. Moreover, an adenosine-based ATP regeneration system was first introduced into E. coli to enhance GSH biosynthesis without exogenous ATP additions. Through the optimization of whole-cell catalytic conditions, the engineered strain GSH17-FDC achieved an impressive GSH titer of 24.19 g/L only after 2 h reaction, with a nearly 100% (98.39%) conversion rate from the added  l-Cys. This work not only unveils a new platform for GSH production but also provides valuable insights for the production of other high-value metabolites that rely on ATP consumption.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Decoupling tourism growth from carbon emissions: Evidence from Chengdu, China.

The fast growth increases in energy use and greenhouse gas emissions in China's tourism industry. This article assessed the carbon emissions of tourist Chengdu's traffic patterns and how they are expected to change between 2005 and 2021 and explains why tourism carbon decoupling is important for sustainable tourism development. In order to investigate how carbon emissions from tourism may expand without necessarily increasing in tandem with GDP and the variables that influence it, the Tapir model and the LMDI (logarithmic mean Divisia index) method were used. In our study, we looked at six important variables, including (1) the number of visitors, (2) the level of tourism spending per capita, (3) the contribution rate of the tourism sector with according to the country's total national income, (4) the amount of passenger traffic relative to GDP, and (5) the energy consumption relative to the volume of passenger traffic. There have been five stages in the correlation: positively charged decoupling, weak decoupling, negative decoupling, strong coupling, and strong decoupling. All of these describe the relationship between the expansion of China's tourist industry and the country's carbon emissions. Findings highlighted the importance of large numbers of visitors, high levels of per-person tourism spending, and low passenger traffic volume per unit of energy consumption as key positive factors. The rise of carbon emissions may also be slowed by increasing the number of passengers transported per unit of GDP. Carbon emissions from tourists in Chengdu are fluctuating, and this influences the city's economy. The findings have significant theoretical and practical implications for Chengdu's transition to a low-carbon economy and for the formulation of policies to reduce emissions. During the research period, most Chinese provinces exhibited ideal decoupling situations. This research has the potential to be used as a scientific resource for guiding the long-term growth as a result of China's tourist sector.

Magnetofluid-integrated biosensors based on DNase-dead Cas12a for visual point-of-care testing of HIV-1 by an up and down chip.

The CRISPR Cas system, as a novel nucleic acid detection tool, is often hindered by cumbersome experimental procedures, complicated reagent transfer processes, and associated aerosol pollution risks. In this study, an integrated nucleic acid detection platform named "up and down chip" was developed, which combined RT-RAA technology for nucleic acid amplification, DNase-dead Cas12a-modified magnetic beads for specific recognition of target nucleic acid, and HRP-TMB chromogenic reaction for signal output in different chambers of a single microfluidic chip. The magnetic beads were migrated in an up-and-down manner between different chambers through magnetic driving, achieving a "sample-in, result-out" detection mode. By introducing a homemade heating box for temperature control during the reaction and using the naked eye or a smartphone APP for color-based signal reading, no professional or precise instruments were required in this platform. Using this platform, highly sensitive detection of the HIV-1 genome as low as 250 copies (CPs) per mL was achieved within 100 min while maintaining good detection performance against common variants as well as excellent specificity and anti-interference ability. In addition, compared with qRT-PCR, it also exhibited good accuracy for 56 spiked plasma samples, indicating its promising potential for clinical application.

Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.

While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.

Integration of a CRISPR Cas12a-assisted multicolor biosensor and a micropipette tip enables visible point-of-care testing of foodborne Vibrio vulnificus.

Foodborne pathogens cause numerous food safety problems, and as a virulent bacterium falling under this category, Vibrio vulnificus (V. vulnificus) poses a huge threat to public health. The conventional methods used for the detection of V. vulnificus, including culture-based and molecular detection methods, have a variety of drawbacks, including being time-consuming and labor-intensive, the requirement of large-scale equipment, and the lack of professional operators. This paper establishes a visible detection platform for V. vulnificus based on CRISPR/Cas12a, which is integrated with nucleic acid isothermal amplification and ß-galactosidase-catalyzed visible color reaction. The specific vvhA gene and a conservative segment in the 16S rDNA gene of the Vibrio genus were selected as the detection targets. By using spectrum analysis, this CRISPR detection platform achieved sensitive detection of V. vulnificus (1 CFU per reaction) with high specificity. Through the color transformation system, as low as 1 CFU per reaction of V. vulnificus in both bacterial solution and artificially contaminated seafood could be visibly observed with the naked eye. Furthermore, the consistency between our assay and the qPCR assay in the detection of V. vulnificus spiked seafood was confirmed. In general, this visible detection platform is user-friendly, accurate, portable, and equipment-free, and is expected to provide a powerful supplement in point-of-care testing of V. vulnificus and also holds good promise for future application in foodborne pathogen detection.

Ecotoxicological effects of soil lithium on earthworm Eisenia fetida: Lethality, bioaccumulation, biomarker responses, and histopathological changes.

Lithium is an emerging environmental contaminant in the current low-carbon economy, but little is known about its influences on soil invertebrates. In this work, earthworm Eisenia fetida was exposed to soils treated with different levels of lithium for 7 d, and multiple ecotoxicological parameters were evaluated. The results showed that mortality was dose-dependent and lithium's median lethal content (LC50) to earthworm was respectively 865.08, 361.01, 139.36, and 94.95 mg/kg after 1 d, 2 d, 4 d, and 7 d exposure. The bioaccumulation factor based on measured exogenous lithium content (BFexog) respectively reached 0.79, 1.01, 1.57, and 1.27 with the increasing lithium levels, suggesting that lithium accumulation was averagely 1.16-fold to the exogenous content, and 74.42%∼81.19%, 14.54%∼18.23%, and 2.26%∼8.02% of the lithium in exposed earthworms were respectively retained in the cytosol, debris, and granule. Then, lithium stress stimulated the activity of superoxide dismutase, peroxidase, catalase, acetylcholinesterase, and glutathione S-transferase as well as the content of 8-hydroxy-2-deoxyguanosine and metallothionein, indicating the generation of oxidative damage, while the content of reactive oxygen species and malondialdehyde decreased. Finally, lithium introduced histopathological changes, including the degenerated seminal vesicle and muscle hyperplasia, as well as high or extreme nuclear DNA damage. This study confirmed the obvious bioaccumulation and toxic effects caused by soil lithium via ecotoxicological data, providing new theoretical insights into understanding the ecological risks of lithium to soil invertebrates.

The greenhouse effect of antibiotics: The influence pathways of antibiotics on methane release from freshwater sediment.

The impact of antibiotics on methane (CH4) release from sediment involves both CH4 production and consumption processes. However, most relevant studies lack a discussion of the pathways by which antibiotics affect CH4 release and do not highlight the role played by the sediment chemical environment in this influence mechanism. Here, we collected field surface sediments and grouped them with various antibiotic combination concentration gradients (50, 100, 500, 1000 ng g-1) under a 35-day indoor anaerobic constant temperature incubation. We found that the positive effect of antibiotics on sediment CH4 release potential appeared later than the positive effect on sediment CH4 release flux. Still, the positive effect of high-concentration antibiotics (500, 1000 ng g-1) occurred with a lag in both processes. Also, the positive effect of high-concentration antibiotics was significantly higher than low-concentration antibiotics (50, 100 ng g-1) in the later incubation period (p < 0.05). We performed a multi-collinearity assessment of sediment biochemical indicators, followed by a generalized linear model with negative binomial regression (GLM-NB) to obtain essential variables. In particular, we conducted the interaction analysis on CH4 release potential and flux regression for the influence pathways construction. The partial least-squares path modeling (PLS-PM) demonstrated that the positive effect of antibiotics on CH4 release (Total effect = 0.2579) was primarily attributed to their effect on the sediment chemical environment (Direct effect = 0.5107). These findings greatly expand our understanding of the antibiotic greenhouse effect in freshwater sediment. Further studies should more carefully consider the effects of antibiotics on the sediment chemical environment, and continuously improve the mechanistic studies of antibiotics on sediment CH4 release.

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per µL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone.

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

AIMS: To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. MATERIALS AND RESULTS: This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. CONCLUSIONS: This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

Dual nucleases-assisted cyclic amplification using polydopamine nanospheres-based biosensors for one-pot detection of microRNAs.

The accurate detection of microRNAs (miRNAs) is essential in the early diagnosis and treatment of cancers. Existing miRNA detection methods represented by nucleic acid amplification (NAA) techniques, such as qRT-PCR, suffer from the small size of miRNAs and lead to limited practicability. CRISPR Cas13a system, another valuable toolbox for nucleic acid detection, relies heavily on the behaviors of accompanying isothermal NAA techniques, which prompts similar deficiencies in miRNA detection. In this study, a dual nucleases-assisted cyclic amplification (DUNCAN) strategy has been established to replace NAA techniques for one-pot detection of miRNAs. The DUNCAN strategy contained an initial reaction based on CRISPR Cas13a for target recognition, and an accompanied cyclic reaction using DNA probes protected by polydopamine nanospheres (PDANSs) for signal amplification and result readout. Exemplified by miR-19b, which has been confirmed to be related to several tumors, the quantitative detection through the DUNCAN strategy was achieved in the dynamic range of 10-106 fM, with a calculated detection limit of 1.27 fM. Besides, the DUNCAN strategy presented well selectivity and anti-interference performance for accurate detection of miR-19b in complex miRNA mixtures, different cell lines and clinical samples compared with qRT-PCR. All these performances demonstrated the promising potential of the DUNCAN strategy in clinical miRNA detection and diagnosis.

Influences of lithium on soil properties and enzyme activities.

Lithium is an emerging environmental contaminant under the current sustainable energy strategy, but little is known about its contamination characteristic in soil. In this study, soil properties and enzyme activities in soils treated with 10-1280 mg kg-1 lithium were measured. The results showed that the content of ammonium nitrogen, total nitrogen, and exchangeable potassium significantly increased by 64.39%-217.73%, 23.06%-131.86%, and 4.76%-16.10%, while electric conductivity and available phosphorus content in lithium treated soils was respectively as 1.10-fold-13.44-fold and 1.27-fold-6.66-fold comparing to CK value. Soil pH and cation exchange capacity slightly declined and increased, respectively, and there was no significant variation in total organic carbon. However, nitrate nitrogen and sulfate content significantly decreased under higher lithium stress. On the other hand, lower lithium treatment level of 10, 20, 40, or 80 mg kg-1 selectively promoted the activities of sucrase, urease, aryl sulfatase, and peroxidase, while the protease, neutral phosphatase, phytase, and lipase were significantly inhibited under all lithium levels, indicating a weaken geochemical cycling of carbon, nitrogen, phosphorus, and sulfur. Then, lithium's 10% and 50% ecological dose (ED10 and ED50) was respectively fitted as 21.18 and 1408.67 mg kg-1 basing on Geometric Mean Index. The influences of lithium on soil were adverse. This study provided important insights into understanding the characteristics of lithium contamination, informing risk assessment and guiding remediation.

Development of a Pichia pastoris cell factory for efficient production of germacrene A: a precursor of ß-elemene.

ß-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to ß-elemene by a one-step reaction in vitro. In this study, we constructed an engineered Pichia pastoris cell factory for producing germacrene A. We rerouted the fluxes towards germacrene A biosynthesis through the optimization of the linker sequences between germacrene A synthase (GAS) and farnesyl pyrophosphate synthase (ERG20), overexpression of important pathway genes (i.e., IDI1, tHMG1, and ACS), and multi-copy integration of related expression cassettes. In combination with medium optimization and bioprocess engineering, the final titer of germacrene A in a 1 L fermenter reached 1.9 g/L through fed-batch fermentation. This represents the first report on the production of germacrene A in P. pastoris and demonstrates its advantage in producing terpenoids and other value-added natural products.

High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis.

Soy leghemoglobin is a key food additive that imparts meaty flavor and color to meat analogs. Here, a Pichia pastoris strain capable of high-yield secretory production of functional leghemoglobin was developed through gene dosage optimization and heme pathway consolidation. First, multi-copy integration of LegH expression cassette was achieved via both post-transformational vector amplification and CRISPR/Cas9 mediated genome editing methods. A combination of inducible expression and constitutive expression resulted in the highest production of leghemoglobin. Then, heme biosynthetic pathway was engineered to address challenges in heme depletion and leghemoglobin secretion. Finally, the disruption of ku70 was complemented in engineered P. pastoris strain to enable high-density fermentation in a 10-L bioreactor. These engineering strategies increased the secretion of leghemoglobin by more than 83-fold, whose maximal leghemoglobin titer and heme binding ratio reached as high as 3.5 g/L and 93 %, respectively. This represents the highest secretory production of heme-containing proteins ever reported.

CRISPR Cas12a-based "sweet" biosensor coupled with personal glucose meter readout for the point-of-care testing of Salmonella.

Salmonella is a pathogen that comes from different animal-originated foods and poses a significant threat to human health. The present detection methods for Salmonella are time-consuming and labor-intensive and requires skilled workers and specialized instruments. In this study, the conservative invA was selected as the target gene, and a quantitative detection method for Salmonella with wide availability and user-friendliness was established based on CRISPR Cas12a and a personal glucose meter (PGM). The indirect signal transformation from the original target DNA to the final glucose signal was achieved through RAA, CRISPR Cas12a reaction, enzymic reaction, and glucose signal reading by a PGM (accu-chek type from Roche). This PGMs-CRISPR assay showed a detection sensitivity of Salmonella as low as 5 colony-forming units (CFU)/reaction in either pure culture or artificially contaminated food samples and exhibited specificity between Salmonella isolates and non-Salmonella isolates. Furthermore, quantitative detection of Salmonella in spiked milk samples was also achieved within the range from 1 to 1 × 103 CFU/reaction. Subsequently, the correlation and consistency between the PGMs-CRISPR assay and quantitative polymerase chain reaction (qPCR) in detection of Salmonella in spiked milk samples were achieved. Therefore, a highly sensitive, portable, quantitative, and user-friendly detection method based on CRISPR Cas12a and PGMs was developed in this study for Salmonella detection and identification. PRACTICAL APPLICATION: A sensitive, rapid, user-friendly, and quantitative detection method based on CRISPR Cas12a for Salmonella in food has been developed in this study, which is of great significance to food safety supervision and management.

Characterization and application of a thermophilic Argonaute from archaeon Thermococcus thioreducens.

Prokaryotic Argonaute proteins (pAgos) play an important role in host defense against invading genetic elements. The functional diversities make pAgos very promising in development of novel nucleic acid manipulation tools and attract increasing attentions. Here, we reported the in vitro characterization of an Argonaute protein from archaeon Thermococcus thioreducens (TtrAgo) and its example of application in hepatitis B virus DNA detection. The results showed that TtrAgo functions as a programmable DNA endonuclease by utilizing both short 5'-phosphorylated and 5'-hydroxylated single-stranded DNA guides, and presents high efficiency and accuracy at optimal temperatures ranging from 75°C to 95°C. In addition, TtrAgo also possesses stepwise cleavage activity like PfAgo (Pyrococcus furiosus) and chopping activity toward double-stranded DNA similar to MjAgo (Methanocaldococcus jannaschii). This study increases our understanding of pAgos and expands the Ago-based DNA detection toolbox.

Efficient poly(ß-L-malic acid) production from cassava hydrolysate by cell recycle of Aureobasidium pullulans.

Poly(ß-L-malic acid) (PMLA) is a water-soluble, biodegradable, and biocompatible polymer with broad prospective applications and can be hydrolyzed to produce widely used acidulant L-malic acid. In order to meet an increasing demand of PMLA, we employed two effective cell-recycling strategies to produce PMLA from raw cassava hydrolysate by Aureobasidium pullulans ZD-3d. In fed-batch fermentation with raw cassava hydrolysate, 101.9 g/L PMLA was obtained with the productivity and yield of 0.77 g/L/h and 0.40 g/g, respectively. Further, three times of membrane filtration-based cell recycling fermentation was carried out, with a high productivity and yield of 1.04-1.64 g/L/h and 0.5-0.84 g/g achieved, respectively. While harnessing centrifugation-based cell recycling fermentation for five times, the productivity and yield approached 0.98-1.76 g/L/h and 0.78-0.86 g/g, respectively. To our knowledge, the processes showed the highest average PMLA productivity compared with others using low-cost biomass, which offered efficient and economical alternatives for PMLA production. KEY POINTS: • PMLA production from raw cassava hydrolysate by Aureobasidium pullulans was studied • High PMLA productivity and yield were obtained via two cell recycling strategies • The highest average PMLA productivity from low-cost biomass to date was achieved.

Effects of one-dimensional nanomaterial polyaniline nanorods on earthworm biomarkers and soil enzymes.

Polyaniline nanorods (PANRs) are typical one-dimensional nanomaterials (1D NMs), which are widely used in medicine, batteries and water treatment, etc. Applications of PANRs will eventually enter the soil environment, but their ecotoxicity has been barely reported. Therefore, we measured earthworm biomass, earthworm biomarkers and soil enzymes to investigate the ecotoxicity of PANRs. The result of positive and increasing growth inhibition rates (GIR) showed that PANRs inhibited earthworm growth. As for earthworm biomarkers, PANRs caused a decrease in protein content, indicating that PANRs stress would increase earthworm energy consumption. Except for the 7th day, the activities of SOD, CAT and POD consistently increased, suggesting that PANRs activated the earthworm antioxidant system. The continually augment of MDA content indicated that PANRs stress would cause earthworm lipid damage. Na+-K+-ATPase increased with an excellent dose-time relationship. Differently, cellulase and AChE activities promoted at low concentrations and inhibited at high concentrations. The positive and dose-dependent IBRv2 indicated that the higher the concentrations of PANRs, the greater the ecotoxicity to earthworms. PANRs inhibited the soil enzyme activities such as sucrase, neutral phosphatase, protease and urease, while induced catalase activity in a dose-dependent manner. Earthworm addition reduced catalase activity by 10.74-29.99%, but improved other soil enzymes activities, demonstrating that earthworms played a positive role in regulating soil enzyme activity. GMean and T-SQI consistently increased due to earthworm activity, meaning a higher soil microbial functional diversity. Generally, this study provided data support for future PANRs toxicity studies, but their toxicity mechanisms still need to be further studied.