Bacterial community analysis of swine manure treated with autothermal thermophilic aerobic digestion

Due to the enviornmental problems associated with disposal of livestock sludge, many stabilization studies emphasizing on the sludge volume reduction were performed. However, little is known about the microbial risk present in sludge and its stabilized products. This study microbiologically explored the effects of anaerobic lagoon fermentation (ALF) and autothermal thermophilic aerobic digestion (ATAD) on pathogen-related risk of raw swine manure by using culture-independent 16S rDNA cloning and sequencing methods. In raw swine manure, clones closely related to pathogens such as Dialister pneumosintes, Erysipelothrix rhusiopathiae, Succinivibrioan dextrinosolvens, and Schineria sp. were detected. Meanwhile, in the mesophilic ALF-treated swine manure, bacterial community clones closely related to pathogens such as Schineria sp. and Succinivibrio dextrinosolvens were still detected. Interestingly, the ATAD treatment resulted in no detection of clones closely related to pathogens in the stabilized thermophilic bacterial community, with the predominance of novel Clostridia class populations. These findings support the superiority of ATAD in selectively reducing potential human and animal pathogens compared to ALF, which is a typical manure stabilization method used in livestock farms.     

Illumina sequencing-based analyses of bacterial communities during short-chain fatty-acid production from food waste and sewage sludge fermentation at different pH values

Short-chain fatty acids (SCFAs) can be produced by primary and waste activated sludge anaerobic fermentation. The yield and product spectrum distribution of SCFAs can be significantly affected by different initial pH values. However, most studies have focused on the physical and chemical aspects of SCFA production by waste activated sludge fermentation at different pH values. Information on the bacterial community structures during acidogenic fermentation is limited. In this study, comparisons of the bacterial communities during the co-substrate fermentation of food wastes and sewage sludge at different pH values were performed using the barcoded Illumina paired-end sequencing method. The results showed that different pH environments harbored a characteristic bacterial community, including sequences related to Lactobacillus, Prevotella, Mitsuokella, Treponema, Clostridium, and Ureibacillus. The most abundant bacterial operational taxonomic units in the different pH environments were those related to carbohydrate-degrading bacteria, which are associated with constituents of co-substrate fermentation. Further analyses showed that during organic matter fermentation, a core microbiota composed of Firmicutes, Proteobacteria, and Bacteroidetes existed. Comparison analyses revealed that the bacterial community during fermentation was significantly affected by the pH, and that the diverse product distribution was related to the shift in bacterial communities.     

Fertilization of SRC Willow,I: Biomass Production Response

Short rotation coppice (SRC) willow is often regarded as one of the most promising crops to increase biomass production and thereby meet the growing demand for renewable energy. This study is based on the hypotheses that biomass production of SRC willow responds positively to increasing doses of nitrogen, and that similar biomass production response can be achieved by use of mineral fertilizer, sewage sludge and animal manure. A 2-year experiment was established with the clone Tordis grown on a sandy soil in northern Jutland, Denmark. The experiment included mineral fertilizer, sludge and manure, and treatments of different doses up to 360 kg nitrogen ha^?1. The fertilization led to a modest but significant increase in biomass production. The largest production of 11.9 oven dried tons/ha/year was obtained for the application of 60 kg nitrogen ha^?1 annually. Higher doses did not lead to increased biomass production; in fact, production seemed to decline with increasing fertilization application (not significant). We found no difference in production between different types of fertilizers. The limited response of the fertilization may be caused by a high fertility of the soil due to former agricultural fertilization. The number of sagging shoots increased significantly with increasing nitrogen dose.     

Activity and Composition of the Denitrifying Bacterial Community Respond Differently to Long-Term Fertilization

The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO₃)₂, (NH₄)₂SO₄, and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The nosZ and narG genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of nosZ and restriction fragment length polymorphism of narG, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the nosZ and narG genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH₄)₂SO₄ and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO₃)₂ or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.     

Assessing the impacts of temperature and storage on <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Salmonella</Emphasis>, and <Emphasis Type="Italic">L. monocytogenes</Emphasis> decay in dairy manure

Elevated levels of animal waste-borne pathogen in ambient water is a serious human health issue. Mitigating influx of pathogens from animal waste such as dairy manure to soil and water requires improving our existing knowledge of pathogen reductions in dairy manure treatment methods. This study was conducted to enhance the  understanding of human pathogen decay in liquid dairy manure in anaerobic (AN) and limited aerobic (LA) storage conditions. The decay of three pathogens (Escherichia coli, Salmonella spp., and Listeria monocytogenes) was assessed in bench-scale batch reactors fed with liquid slurry. A series of temperatures (30, 35, 42, and 50 °C) conditions were tested to determine the impacts of temperature on Escherichia coli, Salmonella, and L. monocytogenes decay in AN and LA conditions. Results showed prolonged survival of E. coli compared to Salmonella and L. monocytogenes in both LA and AN environments. Variations in survival among pathogens with temperature and environmental conditions (i.e., LA and AN) indicated the necessity of developing improved dairy manure waste treatment methods for controlling animal waste-borne pathogens. The results of this study will help in improving the current understanding of human pathogen decay in dairy manure for making informed decisions of animal manure treatment by stakeholders.     

Investigation of keratinolytic and non-keratinolytic fungi grown above or below a 1-cm sewage sludge blanket

This study investigated the incidence of keratinolytic and non-keratinolytic fungi grown above or below a 1-cm sewage sludge blanket. The hair baiting method was used. Incubation was carried out at 23 and 37  °C. The number of keratinolytic fungi occurrences below a sludge blanket (anoxic conditions) was almost two times smaller than the number of fungal occurrences above this blanket (oxic conditions). The anoxic conditions did not significantly affect the number of non-keratinolytic fungi. Qualitative differences were also observed. Trichophyton ajelloi with its teleomorph Arthroderma uncinatum and some other fungi were found to prefer oxic conditions. In the case of non-keratinolytic fungi, the most evident differences were observed at 37  °C. Aspergillus fumigatus prevailed above a sludge blanket, while Pseudallescheria boydii was the predominating species below this blanket. The incidence of keratinolytic fungi was dependent on sludge physico-chemical characteristics: mainly on sludge organic matter stabilization (total nitrogen and ammonium nitrogen contents, proteolytic activity and C:N ratio) and hygienization (total and fecal coliforms) factors.     

长期不同施肥方式对麦田杂草群落的影响

以太湖地区农业科学研究所31a的长期肥料定位试验田为材料,分别于2011—2012年小麦苗期、拔节期和收获期进行了杂草群落调查,研究杂草类型与密度的分布、杂草多样性指数的变化,并对杂草种群分布与土壤养分因子进行冗余分析。结果表明:小飞蓬、看麦娘、大巢菜、稻槎菜、通泉草是本地区小麦生长期的主要杂草类型;随着小麦的生长以及氮肥、有机肥的施入,杂草密度呈下降趋势;施入有机肥降低了麦季杂草的群落多样性指数,在小麦生长的不同时期均衡施肥的CNPK处理以及不施肥的C0处理的群落各项多样性指数能维持在一个较高的水平。RDA分析显示土壤氮含量以及有机质含量与第一排序轴相关性大,是对杂草分布影响最大的两个土壤养分因子。太湖地区稻麦两熟制条件下,长期有机无机肥料单一或配合投入可显著影响麦田杂草的群落组成,其中氮肥和有机肥的施入能显著降低杂草密度;土壤养分的差异影响田面杂草密度和优势种群,均衡施肥能降低优势杂草种群的优势地位,抑制其发生危害程度,提高农田生态系统的生产力及稳定性。     

城市化背景下白洋淀入湖营养盐负荷模拟研究

过量的营养盐输入导致白洋淀富营养化程度较高。雄安新区建设伴随着快速城镇化进程,将可能进一步增加入湖营养盐负荷。为摸清新区建设背景下白洋淀入湖营养盐负荷的变化趋势及削减量,基于入湖营养盐负荷模型计算了历史不同时期（1995-2015年）白洋淀入湖营养盐盐负荷,预测了土地利用、农业管理和污水处理系统变化下入湖营养盐负荷的变化趋势。结果表明,白洋淀历史时期入湖总氮和总磷年平均负荷分别为2018 t和313 t,主要来源分别是耕地和畜禽粪便排放。就土地利用变化而言,以2010年为基准,2050年白洋淀入湖总氮和总磷负荷在建设用地快速增长（RAP）情景下增长率最高,分别增长了56%和60%,主要原因在于城镇人口增加,从而增加了畜禽需求量和生活污水排放。就农业管理而言,削减化肥使用有利于降低入湖总氮负荷,控制畜禽粪便排放更有利于入湖总磷负荷的降低。2050年,生活污水将成为白洋淀入湖营养盐的重要来源,因此,在制定未来社会经济发展路径时,应重点提高农村地区生活污水收集率和处理效率。PCLake模型对不同入湖负荷下湖泊营养状态演变的模拟发现,削减入湖总磷负荷是白洋淀水质恢复的关键。白洋淀水质达到III类和IV类标准时,入湖总磷负荷阈值分别为2.1和2.6 mg P m^-2 d^-1。在建设用地快速增长情景下,若使白洋淀水质达到III类标准,入湖总磷负荷应减少131 t。     

Long-Term Survival of Pathogenic and Sanitation Indicator Bacteria in Experimental Biowaste Composts

For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes, Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli. Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetables, and green waste, were composted in laboratory reactors with forced aeration. The physicochemical and microbiological parameters were monitored for 12 weeks during composting. The survival of bacteria over a 3-month period at 25°C was assessed with samples collected after different experimental composting times. Strain survival was also monitored in mature sterilized composts. Nonsterile composts did not support pathogen growth, but survival of seeded pathogens was observed. Salmonella serovar Enteritidis survived in all composts, and longer survival (3 months) was observed in mature composts (8 and 12 weeks of composting). Mature biowaste composts may support long-term survival of Salmonella serovar Enteritidis during storage at room temperature. E. coli and L. monocytogenes survival was observed only in 4-week-old composts and never in older composts. Proper composting may prevent long-term survival of E. coli and L. monocytogenes. These results suggest that like composted sewage sludge or manure, domestic waste composts may support pathogen survival. Survival was not related to the physicochemical characteristics of the composts.     

Human Enteropathogen Load in Activated Sewage Sludge and Corresponding Sewage Sludge End Products

This study demonstrated a significant reduction in the concentrations of Cryptosporidium parvum and Cryptosporidium hominis oocysts, Giardia lamblia cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.     

Microbial Population Structures in Soil Particle Size Fractions of a Long-Term Fertilizer Field Experiment

Soil structure depends on the association between mineral soil particles (sand, silt, and clay) and organic matter, in which aggregates of different size and stability are formed. Although the chemistry of organic materials, total microbial biomass, and different enzyme activities in different soil particle size fractions have been well studied, little information is available on the structure of microbial populations in microhabitats. In this study, topsoil samples of different fertilizer treatments of a long-term field experiment were analyzed. Size fractions of 200 to 63 μm (fine sand fraction), 63 to 2 μm (silt fraction), and 2 to 0.1 μm (clay fraction) were obtained by a combination of low-energy sonication, wet sieving, and repeated centrifugation. Terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes were used to compare bacterial community structures in different particle size fractions. The microbial community structure was significantly affected by particle size, yielding higher diversity of microbes in small size fractions than in coarse size fractions. The higher biomass previously found in silt and clay fractions could be attributed to higher diversity rather than to better colonization of particular species. Low nutrient availability, protozoan grazing, and competition with fungal organisms may have been responsible for reduced diversities in larger size fractions. Furthermore, larger particle sizes were dominated by α-Proteobacteria, whereas high abundance and diversity of bacteria belonging to the Holophaga/Acidobacterium division were found in smaller size fractions. Although very contrasting organic amendments (green manure, animal manure, sewage sludge, and peat) were examined, our results demonstrated that the bacterial community structure was affected to a greater extent by the particle size fraction than by the kind of fertilizer applied. Therefore, our results demonstrate specific microbe-particle associations that are affected to only a small extent by external factors.     

Anaerobic digestion of pig manure mixed with sewage sludge

This experiment explored the possibility of treating pig manure plus sewage sludge by anaerobic digestion, using batch fermentation at 37°C.The results indicated that the digested materials contained a lower level of putrescible matter when compared to the raw materials. Co-digestion of pig manure and sewage sludge at the ratio of 2:1 seemed to give the best results in terms of reducing the organic load and yielding a higher volume of methane.The experiment should be repeated in digestion tanks of a sewage treatment plant, especially for treating pig manure which has been recognized as the major source of water pollution in Hong Kong.     

Analysis of the structure of bacteria communities and detection of resistance genes of quinolones from pharmaceutical wastewater

The aim of the present study was to investigate the distribution of bacteria and detect the presence of quinolone resistance gene (qnrA) and integrons (intI1, intI2) in a habitat polluted by pharmaceutical sewage. The bacteria were isolated by nutrient agar and nutrient broth from waste water and sludge collected from the sewage outfall of a pharmaceutical factory. The bacteria were identified by Gram staining and biochemical tests, and the bacterial community diversity was analyzed by Shannon–Wiener diversity index (H), Pielou evenness index (J) and Simpson’s diversity index (D). The occurrence of qnrA and integrons (intI1, intI2) were detected by Real-time PCR assays. The results showed that 90 strains were isolated from water samples and sludge samples including 22 genera and 26 species. Types of bacteria in water samples contained 18 genera and 20 species, while 13 genera and 14 species were detected in sludge samples. Fifty-five Enterobacteriaceae isolates (61.11 %, 55 of 90) were the predominant bacteria in water and sludge samples. Bacterial species richness and evenness in water samples were higher than in sludge samples. The resistance genes of qnrA and integrons (intI1, intI2) with the total DNA and single isolate plasmid DNA were detected. There were a variety of bacterial species and the presence of qnrA and integrons (intI1, intI2) genes in pharmaceutical wastewater habitats, in which Enterobacteriaceae strains were the dominant bacteria. These results suggested that pharmaceutical wastewater had potential risks to public health.     

Evaluation of sewage sludge, septic waste and sludge compost applications to corn and forage: yields and N, P and K content of crops and soils

This paper presents the data from two years of experiments concerned with the application of aerobically-digested sewage sludge, anaerobic lagoon septic sludge, sewage sludge compost or fertilizer to soils for grass forage and feed corn (Zea mays L.) production at two different sites 45 km from Truro, Nova Scotia. Crop yields, plant tissue and Mehlich-1 extractable soil nutrients were evaluated; 15 elements were analyzed in the plant tissue and nine elements in the soil extracts. This paper describes the results of crop yields, plant N, P and K content and Mehlich-1 extractable P and K. The research demonstrated the fertilizer produced higher yields of grass forage than the sludge and the compost but equivalent to the sludge in corn yields. Forage and corn N, P and K contents, however, varied with treatment, crop and year, while the compost-amended soils were highest in extractable nutrients. Both sludges and the compost, therefore, could be effective sources of N, P and K for crop production. Compared to the conventional fertilizer, the nutrient availability from the organic amendments (especially N and P) was considerably lower than the 50% assumed at the start of the experiment; the sludges however, provided higher nutrient availability than the compost.     

New Method Using Sedimentation and Immunomagnetic Separation for Isolation and Enumeration of Cryptosporidium parvum Oocysts and Giardia lamblia cysts

A new method for the isolation of Cryptosporidium parvum oocysts and Giardia lamblia cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (P < 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (P > 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts.     

Inactivation by Ionizing Radiation of Salmonella enteritidis Serotype montevideo Grown in Composed Sewage Sludge

S. enteritidis ser. montevideo were grown in composted sewage sludge to levels of approximately 10⁹/g. These bacteria were found to be inactivated by ionizing radiation at approximately the same rate (30 krads/log) as Salmonella species in liquid digested sludge.     

Utilization of cattle manure for Torula yeast production from straw hydrolysates

Good growth of Torula (Candida utilis) was obtained from mixtures of anaerobically fermented cattle manure liquor and barley straw acid hydrolysate. Relatively good growth of this yeast was also obtained from mixtures of acid- or alkali-pretreated cattle manure and barley straw acid hydrolysate. However, a significant amount of reducing sugars (30–40%) was usually left unutilized, indicating the need for a better strain of C. utilis capable of utilizing such sugars as galactose and arabinose which are released (in additon to glucose and xylose) from the hemicellulose during the acid hydrolysis of barley straw. These experiments demonstrate the practicability of using these nutrient sources, obtainable from abundant waste materials, to produce useful single cell protein (SCP) products. In particular, an integrated two stage anaerobic—aerobic fermentation process for the co-production of methane fuel gas and SCP offers an economically attractive option.     

Fertilization of SRC Willow,II: Leaching and Element Balances

Short rotation coppice (SRC) willow is an emerging cropping system in focus for production of biomass for energy. To increase production, the willow is commonly fertilized, but studies have shown differing effects of fertilization on biomass production, ranging from almost no response to considerable positive effects. Focus has also been on replacing mineral fertilizer with organic waste products, such as manure and sludge. However, the effect on biomass production and environmental impact of various dosage and types of fertilizer is not well described. Therefore we studied the environmental impacts of different doses of mineral fertilizer, manure and sewage sludge in a commercially grown SRC willow stand. We examined macro nutrient and heavy metal leaching rates and calculated element balances to evaluate the environmental impact. Growth responses were reported in a former paper (Sevel et al. “Fertilization of SRC Willow, I: Biomass Production Response” Bioenergy Research, 2013). Nitrogen leaching was generally low, between 1 and 7 kg N ha^?1 year^?1 when doses of up to 120 kg N ha^?1 year^?1 were applied. Higher doses of 240 and 360 kg N ha^?1 as single applications caused leaching of 66 and 99 kg N ha^?1 year^?1, respectively, indicating N saturation of the system. Previous intensive farming including high doses of fertilizer may be responsible for a high soil N status and the high N leaching rates. However, moderate fertilization input could not compensate P and K exports with the biomass harvest. No elevated leaching of heavy metals was observed for any fertilization treatments and more cadmium than applied with the fertilizer was removed with the biomass from the system.     

Characteristics and nutrient function of intestinal bacterial communities in black soldier fly (Hermetia illucens L.) larvae in livestock manure conversion

The potential utility of black soldier fly larvae (BSFL) to convert animal waste into harvested protein or lipid sources for feeding animal or producing biodiesel provides a new strategy for agricultural waste management. In this study, the taxonomic structure and potential metabolic and nutrient functions of the intestinal bacterial communities of BSFL were investigated in chicken and swine manure conversion systems. Proteobacteria, Firmicutes and Bacteroidetes were the dominant phyla in the BSFL gut in both the swine and chicken manure systems. After the larvae were fed manure, the proportion of Proteobacteria in their gut significantly decreased, while that of Bacteroidetes remarkably increased. Compared with the original intestinal bacterial community, approximately 90 and 109 new genera were observed in the BSFL gut during chicken and swine manure conversion, and at least half of the initial intestinal genera found remained in the gut during manure conversion. This result may be due to the presence of specialized crypts or paunches that promote microbial persistence and bacteria–host interactions. Ten core genera were found in all 21 samples, and the top three phyla among all of the communities in terms of relative abundance were Proteobacteria, Firmicutes and Bacteroidetes. The nutrient elements (OM, TN, TP, TK and CF) of manure may partly affect the succession of gut bacterial communities with one another, while TN and CF are strongly positively correlated with the relative abundance of Providencia. Some bacterial taxa with the reported ability to synthesize amino acids, Rhizobiales, Burkholderia, Bacteroidales, etc., were also observed in the BSFL gut. Functional analysis based on genes showed that intestinal microbes potentially contribute to the nutrition of BSFL and the high-level amino acid metabolism may partly explain the biological mechanisms of protein accumulation in the BSFL body. These results are helpful in understanding the biological mechanisms of high-efficiency nutrient conversion in BSFL associated with intestinal microbes.