免疫增强剂党参对仿刺参肠道菌群结构的影响

采用平板计数法和聚合酶链式反应( PCR)-变性梯度凝胶电泳( DGGE)技术分析免疫增强剂党参对仿刺参( Apostichopus japonicas)肠道菌群结构的影响。将初始体重为(18.00±2.00) g的仿刺参随机分为2组(对照组、试验组),每组6个重复,每个重复12只。对照组投喂海泥、鼠尾藻粉按照1∶1的质量比配制的饵料,试验组饵料中以鼠尾藻粉质量的2%添加党参,连续投喂28 d。结果表明：应用免疫增强剂党参不仅能够显著提高仿刺参的特定生长率( P<0.05),降低其饵料系数(P<0.05),而且能够显著增加仿刺参肠道内容物中异养菌的数量(P<0.05);序列统计分析显示投喂党参后仿刺参肠道细菌优质序列比例显著增加( P<0.05),试验组达97.57%,对照组仅为80.22%;Beta多样性分析反映投喂党参后仿刺参肠道微生态环境发生了变化,其多样性系数范围在14.91%~15.47%、15.47%~16.21%、14.91%~16.21%;丰度分析显示投喂党参后仿刺参肠道内容物中变形菌门( Proteobacteria)和拟杆菌门( Bacteroidetes)丰度提高,疣微菌门( Verrucomicrobia)、放线菌门( Actinobacteria)和厚壁菌门( Firmiaites)丰度降低;聚类分析显示试验组与对照组肠道菌群结构相似性系数为0.97。由此可见,免疫增强剂党参可提高仿刺参的特定生长率,降低饵料系数,增加肠道异养菌数量和优势菌群丰度,优化肠道微生态环境。     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.     

Intestinal Microbiota in Large Yellow Croaker,Larimichthys crocea,at Different Ages

The polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S ribosomal RNA gene was used to investigate bacterial communities in the intestines of large yellow croaker at six different ages (12 d, 18 d, 26 d, 40 d, 3 mo, and 1 yr old) as well as within the corresponding feed and culture water. In addition, Illumina Miseq sequencing was utilized to compare intestinal microbiota between 12‐d‐old and 1‐yr‐old individuals. PCR‐DGGE results revealed that the culture water had the highest bacterial diversity, followed by the feed, while the intestines had the lowest diversity. The intestinal microbiota at six ages changed severely; however, the change did not follow any trend. The large yellow croaker intestines harbored specific bacterial communities that differed from those in both feed and water. Illumina Miseq sequencing results revealed that the diversity of intestinal bacteria in 12‐d‐old fish was higher than that in 1‐yr‐old fish, and the bacterial composition differed significantly between them. γ‐Proteobacteria and Pseudoalteromonas supplied the most abundant phylum and genus in the 12‐d‐old fish intestine. However, in the 1‐yr‐old fish intestine, Firmicutes and Clostridium were the most dominant, respectively. The study may contribute to a better understanding of gut microbiota and dynamics of the large yellow croaker and the relationship with their surrounding environment.     

Characterization of Nitrate-Reducing and Amino Acid–Using Bacteria Prominent in Nitrotoxin-Enriched Equine Cecal Populations

Horses can be at risk for nitropoisoning by consuming plants containing 3-nitro-1-propionic acid or 3-nitro-1-propanol and to a lesser extent by plants containing nitrate. Populations of equine cecal microbes enriched for enhanced rates of 3-nitro-1-propionic acid (NPA) or nitrate metabolism were cultured for NPA- or nitrate-metabolizing bacteria on basal enrichment medium or tryptose soy agar supplemented with either 5-mM NPA or nitrate and under H₂:CO₂ (20:80) as the energy source. After 72 hours, separated colonies picked from plates, or roll tubes were cultured in fresh broth medium for 72 hours and then identified by 16S rRNA gene sequencing. Isolates from the NPA-enriched populations were identified as Streptococcus lutetiensis (five strains), Escherichia coli (two strains), and Sporanaerobacter acetigens (one strain). Strains isolated from nitrate-enriched populations were identified as Escherichia coli (one strain) and Wolinella succinogenes (three strains). None of these strains degraded NPA. Enriched populations of equine cecal microbes, the isolated pure strains and the type strain of Denitrobacterium detoxificans, a competent NPA-metabolizing microbe, were examined using denaturing gradient gel electrophoresis (DGGE). The DGGE analysis indicated that none of the strains in the enriched population of equine cecal microbes were similar to D. detoxificans. However, we report for the first time the isolation of the anaerobic amino acid–using Sporanaerobacter acetigenes from the equine cecum.     

Manipulation of rumen fermentation and ecology of swamp buffalo by coconut oil and garlic powder supplementation

This experiment was conducted to investigate the effect of coconut oil and garlic powder supplementation on digestibility of nutrients, rumen fermentation, rumen ecology, rumen microorganism and methanogen diversity. Four, 3-year old, rumen fistulated swamp buffalo bulls were randomly assigned in a 4 × 4 Latin square design to receive four dietary treatments; 7% coconut oil, 7% coconut oil with 50 g/day of garlic powder, 7% coconut oil with 100 g/day of garlic powder and non-supplemented (control). During the experiment, concentrate was offered at 0.5% of BW while rice straw was given on ad libitum basis. It was found that supplementation of 7% coconut oil had significantly influenced on total DM intake, OM, NDF and ADF digestibilites while supplementation of 7% coconut oil with garlic powder (50 and 100 g/day) were not significantly different when compared with the control. Dietary supplementations did not affect on rumen pH, NH₃–N concentrations. Blood urea nitrogen (BUN) was significantly higher in supplemented groups. Total VFA concentration, proportion of acetate and acetate to propionate ratio were reduced by supplementation. Proportion of propionate was increased (P < 0.05) when supplemented with 7% coconut oil and 7% coconut oil with 100 g/day of garlic powder. Methane production was dramatically reduced (P = 0.005) in supplemented treatments and was 10% reduced in 7% coconut oil supplementation. Amylolytic and proteolytic bacteria were increased (P = 0.007 and P = 0.024) while protozoal population decreased by 68–75% (P < 0.01) by supplementation. Total bacterial population was increased by supplementation while total fungi and total methanogens were not significantly different among treatments. Percentage of cellulolytic bacterial population was not different among treatments. However, dietary supplementation reduced (P < 0.001) percentage of F. succinogenes population. However, methanogen diversity was not changed using PCR–DGGE as technique. Thirteen separate bands were found in the DGGE gel. Based on this study, supplementation with 7% coconut oil plus 100 g/day of garlic powder could be efficiently utilized in the rumen and thus, could provide good fermentation end products and improve rumen ecology for the host swamp buffaloes particularly in reducing 9% methane gas production without changing nutrient digestibilities.     

土壤微生物多样性的分子生态学研究方法

传统的平板培养法分离培养和鉴定土壤微生物只能反映极少数微生物的信息,种类只占土壤微生物种类总数的0.1%～1%,分子生态学方法应用于土壤微生物的鉴定显示出极大的优越性.着重阐述了土壤微生物多样性的研究内容、意义及目前的采用分子生态学的方法研究土壤微生物多样性,尤其以DGGE（denaturing gradient gel electrophoresis）分子生物学技术以及RAPD（Random amplified polymorphic DNA ）随机扩增的多态性分析方法更为精确和快速,为土壤微生物多样性研究提供了一个更加广阔的前景.     

Comparison of Bacterial Communities Associated with Microcrustaceans from the Floodwater of a Paddy Field Microcosm: Estimation Based on DGGE Pattern and Sequence Analyses

Bacterial communities associated with five kinds of microcrustaceans ( Tanycypris sp., Moina sp., Mesocyclops sp., Cypretta sp. and Heterocypris sp.) from the floodwater of a paddy field microcosm were examined by the application of denaturing gradient gel electrophoresis (DGGE) to PCR-amplified 16S rDNA products with universal bacterial primers and by sequencing of characteristic DGGE bands. The number of DGGE bands of the associated bacteria was small, indicating the association of specific bacterial members with the microcrustaceans studied, among which Tanycypris sp. showed the smallest number of bands. Principal component analysis (PCA) demonstrated that the community structure of the associated bacteria could be divided into three groups: Podocopida ( Tanycypris sp., Cypretta sp. and Heterocypris sp.), Moina sp. and Mesocyclops sp., and further analysis separated Tanycypris sp. and Heterocypris sp. into different clusters. The duration of the incubation period affected the bacteria associated with Tanycypris sp., Moina sp. and Cypretta sp. only. Nearly all of the associated bacteria belonged to Gram-negative bacteria, especially the Cytophaga-Flavobacterium-Bacteroides (CFB) group. Closest relatives of the DGGE bands common to three Podocopida and Mesocyclops sp. belonged to an invertebrate endosymbiont.     

Molecular Monitoring of SRB Community Structure and Dynamics in Batch Experiments to Examine the Applicability of in situ Precipitation of Heavy Metals for Groundwater Remediation (15 pp)

Background, Aims and Scope   Sulfate-reducing bacteria (SRB) are known for their capacity to reduce and precipitate heavy metals (HM) as metal sulfides, offering the opportunity to create an in situ reactive zone for the treatment of heavy metal-contaminated groundwater, a process called in situ metal precipitation (ISMP). The applicability of the ISMP technology first has to be investigated at a laboratory scale before going into an on site application. The evaluation and optimization of the ISMP process is facilitated when physical/chemical analysis techniques are combined with molecular tools that specifically monitor the abundance, diversity and dynamics of the indigenous sulfate reducing microbial community. In this study, batch experiments were conducted in order to investigate the feasibility of ISMP as a groundwater remediation strategy for an industrial site contaminated with elevated levels of Zn, Cd, Co and Ni. Methods   The potential of different types of carbon source/ electron donor (lactate, acetate, methanol, ethanol, Hydrogen Release Compound?, molasses) to stimulate the sulfate reduction and metal precipitation activity of the naturally present (or indigenous) SRB community was explored. In addition, the effect of amending vitamin B12 and yeast extract was evaluated. The ISMP process was monitored by combining analytical analyzes of process parameters (SO42&#61485;-concentration, heavy metal concentrations, pH, Eh) with molecular tools such as SRB subgroup and genus specific PCR, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis of clone sequences, based on either the 16S rRNA or the dsr (dissimilatory sulfite reductase) gene. Results and Discussion   The efficiency of different carbon-sources to stimulate the ISMP process followed the order HRC&#61650; 〉 molasses 〉 methanol 〉 lactate 〉 ethanol 〉 acetate. Within 10 weeks, the highest sulfate and metal removal efficiencies ranged from 85% to 99%. Addition of yeast extract boosted the ISMP process, whereas vitamin B12 negligibly affected SRB activity. Analysis of the sulfate reducing population by SRB subgroup and genus specific PCR demonstrated that members of the genus Desulfosporosinus dominated in all batch tests, while 16S rDNA DGGE profiles additionally revealed the presence in the microbial communities of non-sulfate reducing bacteria within the family Clostridium and the &#61541;-proteobacteria. The dsrB-based DGGE profiles allowed us to assess the diversity and dynamics of the sulfate reducing community and added to a better understanding of the effects of different batch conditions on the ISMP process. Remarkably, all dsrB sequences affiliated with the dsrB gene sequence cluster found in Desulfotomaculum, which received their xenologous dsrB gene from the &#61540;-proteobacteria. Conclusions   The batch experiments, which aimed at stimulating the activities of the indigenous SRB communities, demonstrated that these communities were present and that their activities could be used to obtain efficient in situ precipitation of the contaminating heavy metals. This opens the possibility to test this concept in the future as an on site demonstration as part of the groundwater strategy for the heavy metal contaminated site. Although batch setups are suitable for preliminary feasibility studies for ISMP, they do not reflect the in situ situation where sulfate and heavy metal and metalloid polluted groundwater are supplied continuously. A sulfate reducing strain JG32A was isolated from whose 16S rRNA gene affiliated with the genus Desulfosporosinus, while its dsrB gene sequence clustered with Desulfotomaculum dsrB gene sequences, which received their xenologous dsr genes from &#61540;-proteobacteria. Therefore we hypothesize that the batch experiments enrich members of the Desulfosporosinus genus that possess a non-orthologous dsrB gene. Recommendation and Perspective   The next step towards an on site pilot test for ISMP will be the setup of a series of column experiments, with process conditions that are selected based on the above mentioned results. This will allow to define optimal ISMP process conditions and to test its long-term efficacy and sustainability before going into an on site bioremediation application. By applying the described molecular tools together with physical-chemical analyzes, it can be investigated whether the same SRB community is enriched and which type of C-source is most effective in promoting and sustaining its growth and sulfate-reduction activity.     

Combined effect of fertilizer and herbicide applications on the abundance, community structure and performance of the soil methanotrophic community

The use of agrochemicals, such as mineral fertilizers and herbicides in agricultural systems, may affect the potential of soils to act as a sink for methane. Typically, the effect of each agrochemical on soil methane oxidation is investigated separately whereas in the field these agrochemicals are used together to form one comprehensive land management system. Here we report the results of field experiments that assessed the combined effect of multiple fertilizer and herbicide (nicosulfuron, dimethenamide and atrazine) applications on the soil methanotrophic community. Soils treated with organic fertilizer had three times higher methane oxidation rates compared to soils receiving mineral fertilizers. These higher oxidation rates were positively reflected in a significantly enhanced abundance of methanotrophs for the organic fertilized soils. In contrast, herbicide application did not alter significantly the soil methane oxidation rate or the methane-oxidizing population abundance. Subsequently, the methanotrophic community structure was analyzed with group-specific DGGE of 16S rRNA genes. Cluster analysis of the methanotrophic patterns clearly separated the mineral from organically fertilized soils. Less pronounced clustering differentiated between chemical and manual weed control. Furthermore, cluster analysis of the methanotrophic community revealed that soil type was the primary determinant of the community structure. Our results indicate that fertilizer type had the greatest influence on methane oxidizer activity and abundance. Soil type had the most pronounced effect on the microbial community structure.