Microecological insight to fungal structure and key fungal communities regulating nitrogen transformation based on spatial heterogeneity during cow manure composting by multi-angle and multi-aspect analyses.

Composting is the mainstream technology for the treatment of agricultural solid waste, but limited efforts were made to investigate fungal composition and its contributions to nitrogen transformation in different depths of compost. In this study, spatial distributions of fungi were analyzed using high throughput sequencing by multi-angle analyses, and the key fungal communities determining nitrogen transformation were quantified and identified by multi-aspect analyses during cow manure composting. Multi-angle analyses showed that fungal structure, biomarkers and trophic mode composition varied in different layers, revealing that spatial heterogeneity is the distinctive attribute of composting system. Ascomycota and Basidiomycota were dominant phyla during composting, the two phyla peaked in top and bottom layer respectively. At mesophilic stage, Tremellales, and unclassified Ascomycota (order) were biomarkers in top and middle layer respectively, and so were Remersonia, Pyrenochaetopsis, and Wallemia in bottom layer by LEfSe analysis. Based on multi-aspect analyses, Unclassified Dothideomycetes mainly affected NH4+-N transformation both in top (1.2816***) and middle layers (1.1726*). Trichocladium asperum (0.9536***) and Zopfiella (-0.9484***) mainly affected TN transformation in top layer. Guehomyces pullulans (-0.9684**) and Preussia (-1.0508**) regulated NO3--N transformation in middle layer. Thermomyces lanuginosus (0.7127***) and Typhula sp. UW973129 (0.7298***) were the key species promoting TN and C/N transformation in bottom layer, respectively. Interestingly, different fungal communities showed a complex network interaction driving nitrogen transformation, and the abundance of microbial community could be conducive to characterizing nitrogen transformation in the vertical space of composting.

Correlación entre los microorganismos oxidantes del amonio y los factores ambientales durante el compostaje de estiércol de ganado / Correlation between ammonia-oxidizing microorganisms and environmental factors during cattle manure composting

Abstract Cattle manure composting was performed in an aerated vessel. Community structure and diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) techniques targeting the ammonia monooxygenase alpha subunit (amoA) gene and the correlation between AOB and AOA communities and environmental factors was explored. Thirteen (13) AOB sequences were obtained, which were closely related to Nitrosomonas spp., Nitrosomonas eutropha, and Nitrosospira spp. and uncultured bacteria, among which Nitrosomonas spp. were predominant. Excessively high temperature and high ammonium concentration were not favorable for AOB growth. Five AOA sequences, belonging to Candidatus Nitrososphaera gargensis and to an uncultured archaeon, were obtained. During composting, community diversity of AOB and AOA fluctuated, with AOA showing a higher Shannon-Wiener index. The AOB community changed more dramatically in the mesophilic stage and the early thermophilic stage, whereas the most obvious AOA community succession occurred in the late thermophilic stage, the cooling stage and the maturity stage. Water content, total nitrogen (TN) and ammonium concentration were more relevant to the AOB community structure, while higher correlations were observed between ammonia, nitrate and TN and the AOA community. AOB community diversity was negatively correlated with pH (r = -0.938, p < 0.01) and water content (r = -0.765, p < 0.05), while positively correlated with TN (r = 0.894, p < 0.01). AOA community diversity was negatively correlated with ammonium concentration (r = -0.901, p < 0.01). Ammonium concentration played an important role in the succession of AOB and AOA communities during composting.

Resumen Se llevó a cabo un compostaje de estiércol de ganado en un recipiente aireado. Se investigó la estructura de la comunidad y la diversidad de bacterias oxidantes del amoníaco (AOB) y las arqueas oxidantes del amoníaco (AOA) mediante el uso de las técnicas de reacción en cadena de la polimerasa y la electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) dirigidas al gen de la subunidad alfa de la amonio monooxigenasa (amoA), y se exploró la correlación entre las comunidades AOB, AOA y los factores ambientales. Se obtuvieron 13 secuencias de AOB, las cuales se relacionaron estrechamente con Nitrosomonas spp., Nitrosomonas eutropha y Nitrosospira spp., y bacterias no cultivadas, entre las cuales fueron predominantes las Nitrosomonas spp. La temperatura excesivamente alta y la concentración de amonio elevada no fueron favorables para el crecimiento de las AOB. Se obtuvieron 5 secuencias de AOA, pertenecientes a Candidatus Nitrososphaera gargensis y un Archaeon no cultivado. Durante el compostaje, la diversidad de AOB y AOA fluctuó y las AOA mostraron un índice de Shannon-Wiener más alto. La comunidad de AOB cambió significativamente en la etapa mesofílica y la etapa termofílica temprana, mientras que la sucesión más obvia de la comunidad AOA ocurrió en la etapa termofílica tardía y las etapas de enfriamiento y de maduración. El contenido de agua, el nitrógeno total (TN) y la concentración de amonio fueron más relevantes para la estructura de la comunidad AOB, mientras que se observaron correlaciones mayores entre amoníaco, nitrato y TN, y la comunidad AOA. La diversidad de la comunidad AOB se correlacionó negativamente con el pH (r= -0,938; p < 0,01) y el contenido de agua (r = -0,765; p < 0,05), mientras que se relacionó positivamente con TN (r = 0,894; p < 0,01). La diversidad de la comunidad AOA se correlacionó negativamente con la concentración de amonio (r = -0,901; p < 0,01). La concentración de amonio desempenó un papel importante en la sucesión de las comunidades AOB y AOA durante el compostaje.

Diversity and abundance of denitrifiers during cow manure composting / Diversidad y abundancia de desnitrificadores durante el compostaje del estiércol de vaca

Diversity and abundance of the denitrifying genes nirK, nirS and nosZ were investigated in cow manure compost using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time quantitative PCR (qPCR), respectively. These three genes were detected in all the stages of the composting process. Phylogenetic analysis showed that the nirK gene was closely related to Rhizobiales, Burkholderiales, the nirS gene was closely related to Pseudomonadales and Burkholderiales, and the nosZ gene was closely related to Rhodospirillales, Rhizobiales, Pseudomonadales, and Alteromonadales. qPCR results showed that the abundance of these three genes (nirK, nirS and nosZ) reached the peak value in the late thermophilic stage of composting and abundance of the nirK gene was higher than that of the nosZ gene and the nirS gene. Redundancy analysis (RDA) showed that the diversity of the nirK and nirS genes was significantly correlated with ammonium (p < 0.05), the diversity of the nosZ gene was significantly correlated with pH (p < 0.05) and the abundance of the nirK nirS and nosZ genes was significantly correlated with temperature (p< 0.05).

La diversidad y la abundancia de los genes desnitrificadores nirK, nirS, nosZ en el compost de estiércol de vaca se investigaron por medio de la reacción en cadena de la polimerasa seguida de electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) y por PCR cuantitativa (qPCR) en tiempo real, respectivamente. Estos 3 genes fueron detectados durante todas las fases del compostaje. El análisis filogenético mostró estrecha relación del gen nirK con Rhizobiales y Burkholderiales, del gen nirS con Pseudomonadales y Burkholderiales y del gen nosZ con Rhodospirillales, Rhizobiales, Pseudomonadales y Alteromonadales. Los resultados de la qPCR mostraron que la abundancia de los genes nirK, nirSy nosZ alcanzó el valor máximo en la fase termofílica tardía del compostaje, y que la abundancia del gen nirK era más elevada que los de los genes nosZ y nirS. El análisis de redundancia (RDA) mostró que la diversidad de los genes nirK y nirS estaba significativamente correlacionada con la concentración de amonio (p<0,05), mientras que la del gen nosZ estaba significativamente correlacionada con el pH (p<0,05). También mostró que la abundancia de los genes nirK, nirS y nosZ estaba significativamente correlacionada con la temperatura (p<0,05).

Assessing key microbial communities determining nitrogen transformation in composting of cow manure using illumina high-throughput sequencing.

Insight to nitrogen transformation and cycling during composting is vital in developing management strategies that improve nitrogen content and quality of the end product. In this study, a positive ventilation device was constructed and used to elucidate nitrogen transformation and microbial community structures during the composting of cow manure and rice straw. Bacterial community successions were analyzed during the composting process by examining the change in their structural dynamics using high-throughput sequencing technique. The results revealed that dominant phyla, included Acidobacter, Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Actinobacteria. Furthermore, a positive strong correlation was observed between the key bacterial communities and nitrogen transformation. Analyses of functional genera, Spearman correlation and Path showed that Thermomonospora_curvata_DSM_43183 followed by Luteimonas and Simiduia, Brevundimonas and Tamlana, Pseudomonas followed by Brevundimonas and Flavobacterium were the key bacterial communities affecting NH4+-N, NO3--N, and NO2--N transformation, respectively. Thauera followed by Pseudomonas_putida_NBRC_14164 played a dominant role in N2O transformation.

Microbial Community Succession and Response to Environmental Variables During Cow Manure and Corn Straw Composting.

In composting system, the composition of microbial communities is determined by the constant change in the physicochemical parameters. This study explored the dynamics of bacterial and fungal communities during cow manure and corn straw composting using high throughput sequencing technology. The relationships between physicochemical parameters and microbial community composition and abundance were also evaluated. The sequencing results revealed the major phyla included Proteobacteria, Bacteroidetes, Firmicutes, Chloroflexi and Actinobacteria, Ascomycota, and Basidiomycota. Linear discriminant analysis effect size (LEfSe) illustrated that Actinomycetales and Sordariomycetes were the indicators of bacteria and fungi in the maturation phase, respectively. Mantel test showed that NO3 --N, NH4 +-N, TN, C/N, temperature and moisture content significantly influenced bacterial community composition while only TN and moisture content had a significant effect on fungal community structure. Structural equation model (SEM) indicated that TN, NH4 +-N, NO3 --N and pH had a significant effect on fungal abundance while TN and temperature significantly affected bacterial abundance. Our finding increases the understanding of microbial community succession in cow manure and corn straw composting under natural conditions.

Bacterial community succession in dairy manure composting with a static composting technique.

This study applied high-throughput sequencing technology and PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved state) to examine the microbial population dynamics during the composting of dairy manure and rice straw in a static (without turning) composting system. The results showed that the composition of the bacterial community varied significantly during the composting process. The dominant phyla included Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Chloroflexi. Biodiversity indices showed that bacterial community diversity had the peak value during the mesophilic phase. Redundancy analysis indicated that nitrogen was the most important factor in the distribution of genera during the composting process. Finally, the Pearson correlation results suggested that Thermomonospora and Thermopolyspora could be the biomarkers of the composting maturation phase. The metabolic characteristics of the bacterial communities were predicted by PICRUSt. The result showed that metabolism of amino acids, lipids, and most of the carbohydrates increased during the whole composting treatment. However, methane metabolism, carbon fixation pathways in prokaryotes, and nucleotide metabolism decreased after the thermophilic phase. The present study provides a better understanding for bacterial community composition and function succession in dairy manure composting.

Correlation between ammonia-oxidizing microorganisms and environmental factors during cattle manure composting.

Cattle manure composting was performed in an aerated vessel. Community structure and diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) techniques targeting the ammonia monooxygenase alpha subunit (amoA) gene and the correlation between AOB and AOA communities and environmental factors was explored. Thirteen (13) AOB sequences were obtained, which were closely related to Nitrosomonas spp., Nitrosomonas eutropha, and Nitrosospira spp. and uncultured bacteria, among which Nitrosomonas spp. were predominant. Excessively high temperature and high ammonium concentration were not favorable for AOB growth. Five AOA sequences, belonging to CandidatusNitrososphaera gargensis and to an uncultured archaeon, were obtained. During composting, community diversity of AOB and AOA fluctuated, with AOA showing a higher Shannon-Wiener index. The AOB community changed more dramatically in the mesophilic stage and the early thermophilic stage, whereas the most obvious AOA community succession occurred in the late thermophilic stage, the cooling stage and the maturity stage. Water content, total nitrogen (TN) and ammonium concentration were more relevant to the AOB community structure, while higher correlations were observed between ammonia, nitrate and TN and the AOA community. AOB community diversity was negatively correlated with pH (r = -0.938, p < 0.01) and water content (r = -0.765, p < 0.05), while positively correlated with TN (r = 0.894, p < 0.01). AOA community diversity was negatively correlated with ammonium concentration (r = -0.901, p < 0.01). Ammonium concentration played an important role in the succession of AOB and AOA communities during composting.

Diversity and abundance of denitrifiers during cow manure composting.

Diversity and abundance of the denitrifying genes nirK, nirS and nosZ were investigated in cow manure compost using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time quantitative PCR (qPCR), respectively. These three genes were detected in all the stages of the composting process. Phylogenetic analysis showed that the nirK gene was closely related to Rhizobiales, Burkholderiales, the nirS gene was closely related to Pseudomonadales and Burkholderiales, and the nosZ gene was closely related to Rhodospirillales, Rhizobiales, Pseudomonadales, and Alteromonadales. qPCR results showed that the abundance of these three genes (nirK, nirS and nosZ) reached the peak value in the late thermophilic stage of composting and abundance of the nirK gene was higher than that of the nosZ gene and the nirS gene. Redundancy analysis (RDA) showed that the diversity of the nirK and nirS genes was significantly correlated with ammonium (p<0.05), the diversity of the nosZ gene was significantly correlated with pH (p<0.05) and the abundance of the nirK nirS and nosZ genes was significantly correlated with temperature (p<0.05).

Aliibacillus thermotolerans gen. nov., sp. nov.: a thermophilic and heterotrophic ammonia-oxidizing bacterium from compost.

A novel moderately thermophilic and heterotrophic ammonia-oxidizing bacterium, designated strain BM62T, was isolated from compost in the thermophilic stage in Harbin, China. Phylogenetic analysis based on the 16S rRNA gene indicated that strain BM62T belongs to the family Bacillaceae within the class Bacilli and was most closely related to Alteribacillus iranensis X5BT (only 94.6% sequence similarity). Cells of strain BM62T were Gram-positive, rod-shaped, motile by periflagella, catalase-positive and oxidase-negative. Growth of strain BM62T was observed at salinities of 0-4% (optimum 2-3%), temperatures of 35-65 °C (optimum 50 °C) and pH values of 5-9 (optimum pH 7). The major cellular fatty acid was iso-C16:0, and the predominant ubiquinone was MK-7. The peptidoglycan type is A1γ, and meso-diaminopimelic acid was the diagnostic diamino acid. The major polar lipids were diphosphatidylglycerol, phospholipid and phosphatidylglycerol. The G + C content of its genomic DNA was 36.5 mol%. Data from this polyphasic taxonomy study suggested that strain BM62T should be classified as the type strain of the type species of a new genus within the family Bacillaceae for which the name Aliibacillus thermotolerans gen. nov., sp. nov. is proposed. The type strain of the species Aliibacillus thermotolerans sp. nov. is BM62T (= DSM 101851T = CGMCC 1.15790T). The respective DPD Taxon Number is GA00057.