Different rapid startups for high-solid anaerobic digestion treating pig manure: Metagenomic insights into antibiotic resistance genes fate and microbial metabolic pathway.

High-solid anaerobic digestion (HSAD), as an emerging disposal technology for swine manure, was commonly hampered by the long lag phase and slow startup, resulting in poor performance. Rapid startups by different leachate reflux forms can solve the problem, but related study was scarcely reported. Therefore, metagenomic analysis was used to exploit the effects of different rapid startups on the biogas performance, antibiotic resistance genes (ARGs) removal and microbial metabolic pathway during HSAD. Compared anaerobic digestion with natural start (T1), three different rapid startups were set, including with autologous leachate reflux (T2), with water reflux (T3) and with exogenous leachate reflux (T4). The results showed that rapid startups (T2-T4) enhanced biogas yield and the cumulative methane yield was increased by 3.7-7.3 times compared with the control. Totally, 922 ARGs were found, most of which belonged to multidrug and MLS ARGs. About 56% of these ARGs could be reduced in T4, while just 32% of ARGs were reduced in T1. Antibiotic efflux pump is the main mechanism of microbial action, which could be decreased largely by these treatments. Moreover, all the rapid startups (T2-T4) made Methanosarcina content (9.59%-75.91%) higher than that in the natural startup of T1 (4.54%-40.27%). This is why these fast-startups helped methane production fast. Network analysis showed that microbial community and environmental factors (pH and VFAs) both contributed to the spread of ARGs. The reconstructed methane metabolic pathway by different identified genes showed that all methanogenesis pathways existed but acetate metabolic pathway was dominant. And the rapid startups made the abundance of acetate metabolic (M00357) higher than the natural startup.

Exploring antibiotic resistance load in paddy-upland rotation fields amended with commercial organic and chemical/slow release fertilizer.

Agricultural fertilization caused the dissemination of antibiotic resistance genes (ARGs) in agro-ecological environment, which poses a global threat to crop-food safety and human health. However, few studies are known about the influence of different agricultural fertilization modes on antibiotic resistome in the paddy-upland rotation soils. Therefore, we conducted a field experiment to compare the effect of different fertilization (chemical fertilizer, slow release fertilizer and commercial organic fertilizer replacement at various rates) on soil antibiotic resistome in paddy-upland rotation fields. Results revealed that a total of 100 ARG subtypes and 9 mobile genetic elements (MGEs) occurred in paddy-upland rotation soil, among which MDR-ARGs, MLSB-ARGs and tet-ARGs were the dominant resistance determinants. Long-term agricultural fertilization remarkably facilitated the vertical accumulation of ARGs, in particular that bla ampC and tetO in relative abundance showed significant enrichment with increasing depth. It's worth noting that slow release fertilizer significantly increased soil ARGs, when comparable to manure with 20% replacing amount, but chemical fertilizer had only slight impact on soil ARGs. Fertilization modes affected soil microbial communities, mainly concentrated in the surface layer, while the proportion of Proteobacteria with the highest abundance decreased gradually with increasing depth. Furthermore, microbial community and MGEs were further proved to be essential factors in regulating the variability of ARGs of different fertilization modes by structural equation model, and had strong direct influence (λ = 0.61, p < 0.05; λ = 0. 55, p < 0.01). The results provided scientific guidance for reducing the spreading risk of ARGs and control ARG dissemination in agricultural fertilization.

Effects of initial volatile fatty acid concentrations on process characteristics, microbial communities, and metabolic pathways on solid-state anaerobic digestion.

Solid-state anaerobic digestion (SSAD) is vulnerable to excess volatile fatty acids (VFA), mainly acetate and propionate. The co-effects of VFAs and microbial dynamics under VFA accumulation were investigated in SSAD of pig manure and corn straw. Adding 2 and 4 mg/g acetate or propionate caused initial increases in total VFAs, followed by decreases after day 6, resulting in 'mild' VFA accumulation, while adding 6 mg/g caused similarly increased VFAs, but with no subsequent decrease, causing 'severe' VFA accumulation and poor methanation performance. Mild propionate accumulation promoted acetate consumption, whereas acetate accumulation inhibited propionate degradation by affecting crucial redox reactions. Under severe VFA accumulation, hydrolysis and acidification mainly conducted by acid-tolerant Clostridium sp. exacerbated VFA inhibition, causing a competition between Methanosarcina and Methanosaeta, and impairments of acetoclastic and hydrogenotrophic methanogenesis and interspecies formate transfer. This study provides new insights into mechanisms of VFA accumulation in SSAD, and its effects on methanogenesis.

Deciphering discriminative antibiotic resistance genes and pathogens in agricultural soil following chemical and organic fertilizer.

Fertilizers containing rich nutrients can change the profiles of antibiotic resistant pathogens (ARPs) and antibiotic resistance genes (ARGs) in receiving soils; however, the discriminative ARGs and ARPs in agricultural soil following different fertilizer applications remain unknown. Using metagenomic sequencing combined with binning approach, the present study investigated the discriminative ARGs and ARPs under various fertilizer applications (chemical and organic fertilizer) in a 8-year field experiment. VanR, multidrug ARG transporter, vanS, ermA, and arnA were the discriminative ARGs in the chemical fertilizer group, whereas rosB, multidrug transporter, mexW, and aac(3)-I were enhanced in the organic fertilizer group. The metagenomic binning approach revealed that both fertilizer applications caused pathogen proliferation. Chemical fertilizer caused the increase in the pathogenic genus Luteimonas, and organic fertilizer facilitated the proliferation of the pathogenic genera Dokdonella and Pseudomonas. The pathogenic species Pseudomonas_H sp014836765, carrying mexW and multidrug transporter, was enriched only in the organic fertilizer group, indicating that it was a discriminative ARP in the organic fertilizer group. Our results demonstrated that fertilizer application, particularly organic fertilizer application, can facilitate the proliferation of ARGs and ARPs in the receiving soil, posing the risk of the development and spread of soil-borne ARPs.

Investigating changes in the characteristics of antibiotic resistance genes at different reaction stages of high solid anaerobic digestion with pig manure.

Contamination of antibiotic resistance genes (ARGs) from animals is a serious issue as they may spread to human pathogenic bacteria. The reduction of ARG contamination from livestock waste is thus essential. High solid anaerobic digestion (HSAD) is a new and effective technology although some aspects, such as change characteristics of ARGs at different reaction stages, have not been fully investigated. This study focused firstly on the variations in ARGs at different reaction stages in HSAD systems with five different starting methods: 1 natural start (the control) and 4 rapid starts by changing leachate reflux forms. The results showed that the rapid starting methods could accelerate start-up and increase biogas production by 312.5%. The starting and acidification stages showed higher microbial richness and diversity compared with the other stages. ARGs found to be reduced at acidification stage. Variation in ARGs at the starting and acidification stages was mainly driven by a combination of microbial community, mobile genetic elements (MGEs), and environmental factors; while the main contributory factors at the gas production stage were biomass and several unexplained factors. At the ending stage, the main driving factors were biomass and microbial communities. Most of the potential hosts (16/20) of the ARGs belonged to the Firmicutes phylum, which showed the lowest connections with the ARGs at the gas production stage.

Long-term urbanization impacts the eastern golden frog (Pelophylax plancyi) in Shanghai City: Demographic history, genetic structure, and implications for amphibian conservation in intensively urbanizing environments.

Understanding the mechanisms of how urbanization influences the evolution of native species is vital for urban wildlife ecology and conservation in the Anthropocene. With thousands of years of agriculture-dominated historical urbanization followed by 40 years of intensive and rapid urbanization, Shanghai provides an ideal environment to study how the two-stage urbanization process influences the evolution of indigenous wildlife, especially of anuran species. Therefore, in this study, we used mitochondrial Cyt-b gene, microsatellite (SSR), and single nucleotide polymorphism (SNP) data to evaluate the demographic history and genetic structure of the eastern golden frog (Pelophylax plancyi), by sampling 407 individuals from 15 local populations across Shanghai, China. All local populations experienced bottlenecks during historical urbanization, while the local populations in urban areas maintained comparable contemporary effective population sizes (N e) and genetic diversity with suburban and rural populations. Nevertheless, the rapid modern urbanization has already imposed significant negative effects to the integrity of populations. The 15 local populations were differentiated into eight genetic clusters, showing a spatial distribution pattern consistent with the current urbanization gradient and island-mainland geography. Although moderate gene flow still occurred from the rural peripheral cluster to urban and suburban clusters, population fragmentation was more serious in the urban and suburban populations, where higher urbanization levels within 2-km radius areas showed significant negative relationships to the N e and genetic diversity of local populations. Therefore, to protect urban wildlife with limited dispersal ability, improving conditions in fragmented habitat remnants might be most essential for local populations living in more urbanized areas. Meanwhile, we highlight the need to preserve large unfragmented rural habitats and to construct corridor networks to connect discrete urban habitat remnants for the long-term wildlife conservation in intensively urbanizing environments.

Comparative genome analysis of multidrug-resistant and susceptible Glaesserella parasuis strains isolated from diseased pigs in China.

Glaesserella parasuis (G. parasuis) is a respiratory pathogen of swine and the etiological agent of Glässer's disease. Although the emergence of multidrug-resistant (MDR) G. parasuis is a critical problem in the swine industry, there are few publications on the genetic basis of antimicrobial resistance of G. parasuis. In this study, comparative genome analyses were used to identify genomic differences between two phenotypically distinct isolates, an MDR isolate (HPS-1) and a susceptible isolate (HPS-2), from diseased swines in China. These isolates were both serovar 4, which is predominant in cases of Glässer's disease and is the most prevalent serovar in China. Based on clusters of orthologous group (COG) annotations, genes assigned to the extracellular structure category were only detected in HPS-1 and genes related to cell motility were more abundant in HPS-1 than in HPS-2. A comparative genomic analysis showed that these two isolates are closely related, although there was a large-scale genomic rearrangement. Eighteen percent of the genome consisted of strain-specific accessory genes of HPS-1. Notably, only the two genes aac(6')-Ie-aph(2'')-Ia and blaROB-1 on a plasmid were specific to HPS-1. We also detected 30,599 single nucleotide polymorphisms (SNPs), including nonsynonymous SNPs in the aminoglycoside resistance gene aph(3'')-Ib, the fusidic acid resistance gene fusA, and the two rifampicin resistance genes rpoC and rpoB in HPS-1. These findings improve our understanding of the differences between MDR and susceptible isolates and will aid the development of treatment strategies to decrease the prevalence and disease burden caused by G. parasuis.

Methanogenic community during the anaerobic digestion of different substrates and organic loading rates.

Three anaerobic reactors using pig manure (PM), maize straw (MS), and a mixture of the two as substrates were compared for archaeal community structure and diversity, and for methanogens response to increased organic loading rate (OLR, expressed in the mass of volatile solid (VS)). Methanogenic archaeal richness during codigestion of pig manure with maize straw (ACE: 2412) was greater than that during the others (ACE: 1225, 1467) at an OLR of 4 g L-1  day-1 , accompanied by high specific methane yield. Euryarchaeota and Crenarchaeota predominated during overall digestion of different substrates; with relative abundances of 63.5%-99.0% and 1.0%-36.3%, respectively. Methanosarcina was the predominant genus that accounted for 33.7%-79.8% of the archaeal community. The diversity in the PM digester decreased with increase in OLR, but increased in the MS digester. The diversity was stable during the codigestion with increased OLR. The relative abundances of hydrogenotrophic methanogens increased by 2.6 and 2.1 folds; the methanogenic community shifted from acetoclastic to hydrogenotrophic methanogens during digestion of MS, and of the mixture of MS and PM. Canonical correspondence analysis revealed a strong relationship between reactor parameters and methanogenic community.

Diverse patterns of genomic targeting by transcriptional regulators in Drosophila melanogaster.

Annotation of regulatory elements and identification of the transcription-related factors (TRFs) targeting these elements are key steps in understanding how cells interpret their genetic blueprint and their environment during development, and how that process goes awry in the case of disease. One goal of the modENCODE (model organism ENCyclopedia of DNA Elements) Project is to survey a diverse sampling of TRFs, both DNA-binding and non-DNA-binding factors, to provide a framework for the subsequent study of the mechanisms by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TRFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also highlight the stringency of the Polycomb regulatory network, and show association of the Trithorax-like (Trl) protein with hotspots of DNA binding throughout development. Furthermore, the data identify more than 5800 instances in which TRFs target DNA regions with demonstrated enhancer activity. Regions of high TRF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TRF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. Together these data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development.

Mechanisms of protein kinase Sch9 regulating Bcy1 in Saccharomyces cerevisiae.

In this study, we investigated the mechanisms of Sch9 regulating the localization and phosphorylation of Bcy1. Our research indicated that Sch9 regulated localization of Bcy1 via Zds1 for the following reasons: (1) deletions of SCH9 or ZDS1 both caused nuclear localization of Bcy1; (2) Sch9 and Zds1 interacted physically; (3) overexpression of ZDS1 led to a significantly increased cytoplasmic localization of Bcy1 in sch9Δ cells, whereas overexpression of SCH9 had no visible effect on cytoplasmic localization of Bcy1 in zds1Δ cells. Our study also suggested that Sch9 regulated phosphorylation of Bcy1 via Yak1.

Role of Sch9 in regulating Ras-cAMP signal pathway in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae PKA plays a major role in regulating cell growth, metabolism, and stress resistance. We report that Sch9 regulates PKA directly and SCH9 deletion enhances PKA activity by showing that: (1) Bcy1 predominately localized in the nucleus in glycerol-grown sch9Δ cells; (2) large part of the catalytic subunits of PKA transferred from the nucleus to the cytoplasm in sch9Δ cells; (3) higher protein stability of Tpk2 resulted in higher protein level of Tpk2 in sch9Δ than in wild type cells. Our investigations suggest that Sch9 regulates phosphorylation of Bcy1. We also observed hyper-phosphorylation of Cdc25 in sch9Δ, in contrast to the tpk2Δ and tpk2Δsch9Δ mutants, suggesting that feedback inhibition of PKA on Cdc25 is through Tpk2.