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1.
Seasonal studies of surface sediment bacterial communities, from two basins with differing trophic states within Lake Balaton (Hungary), were carried out using molecular (denaturing gradient gel electrophoresis, DGGE) and cultivation-based techniques. The presence of polyphosphate accumulates was tested using Neisser staining, and phosphatase activity was investigated on organic phosphorus (P) compound. Aerobic viable cell counts were significantly higher in the eutrophic than mesotrophic basin in each season. The lowest viable counts were observed in the autumn and the highest in spring and summer month in both basins. The DGGE fingerprints of the samples reflected that the composition of sediment bacterial communities in the two basins were distinct in spring and summer, and similar in autumn, but similarly diverse in all seasons. On the basis of partial 16S rDNA sequences, the 216 strains were affiliated with six major bacterial lineages: Firmicutes; Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Common species characterized from both basins constituted up to 66% of all identified phylotypes. Strains related to Bacillus sp. were dominant in all but one sample. Isolates affiliated with Aeromonas sp. prevailed in the sample taken from the mesotrophic basin in spring. The majority of the strains showed excess poly-P accumulation. Association of Neisser staining and phosphatase activity test results suggested that excess poly-P accumulation serves as P storage for sediment bacteria. Our study implied the importance of Firmicutes, Actinobacteria, Alphaproteobacteria, and Aeromonas species in benthic bacterial P retention.  相似文献   

2.
典型高原湖滨带底泥细菌群落结构及多样性特征   总被引:5,自引:1,他引:4  
【背景】高原湖泊的富营养化日趋严重,而湖滨带作为湖泊的保护屏障对外源污染物具有拦截净化等作用,水环境变化则会对底泥细菌产生深刻影响。【目的】探究高原湖滨带底泥细菌群落结构特征及与水体富营养化之间的联系。【方法】基于16S rRNA基因高通量测序技术分析了阳宗海南岸湖滨带8个不同样点的底泥细菌群落结构及多样性,并结合样品水体环境因子,采用主成分分析(PCA)和冗余分析(redundancy analysis,RDA)探讨了水体富营养化对底泥细菌群落结构及丰富度的影响。【结果】湖滨带底泥细菌与水体富营养化程度存在响应关系,在水体富营养化程度高的区域(S3)细菌丰富度较高,操作分类单元(operationaltaxonomicunits,OTU)高达1473。反之,在富营养化程度低的区域(S1)细菌丰富度较低,OTU为730。阳宗海南岸湖滨带底泥中主要优势菌门为变形菌门(Proteobacteria)和绿弯菌门(Chloroflexi),含有少量的放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)和厚壁菌门(Firmicutes);绿弯菌门(Chloroflexi)与水体富营养化程度具有相关性,在中度富营养化区域,绿弯菌门(Chloroflexi)的比重高达44.1%,而在轻度富营养化区域绿弯菌门(Chloroflexi)的比重仅为15.6%。通过环境因子分析发现,阳宗海湖滨底泥细菌受总磷(TP)、叶绿素a (Chla)和总氮(TN)影响较强。【结论】研究结果明确了高原湖泊湖滨带底泥细菌种群的结构、变化特征及其对于水体富营养化的响应,加深了高原湖泊底泥细菌的了解,为高原湖泊水体富营养化的防治提供理论基础。  相似文献   

3.
Spatial and temporal variations in sediment microbial community structure in a eutrophic lake polluted with inorganic mercury were identified using polar lipid fatty acid (PLFA) analysis. Microbial community structure was strongly related to mercury methylation potential, sediment organic carbon content, and lake location. Pore water sulfate, total mercury concentrations, and organic matter C/N ratios showed no relationships with microbial community structure. Seasonal changes and changes potentially attributable to temperature regulation of bacterial membranes were detectable but were less important influences on sediment PLFA composition than were differences due to lake sampling location. Analysis of biomarker PLFAs characteristic of Desulfobacter and Desulfovibrio groups of sulfate-reducing bacteria suggests that Desulfobacter-like organisms are important mercury methylators in the sediments, especially in the Lower Arm of Clear Lake.  相似文献   

4.
G. Palijan  D. Fuks 《Hydrobiologia》2006,560(1):405-415
The purpose of this investigation was to determine the influence of different phases of the hydrological cycle on the abundance of colony-forming units (CFU) of bacteria in the Kopački Rit floodplain and to assess temporal and spatial variations in the proportion of eutrophic and oligotrophic bacteria (r- and K-strategists) as a biological indicator of dissolved organic carbon (DOC) availability. Determination of bacterial abundance was performed with abiotic variables (water temperature, depth, Secchi disc transparency, dissolved oxygen, pH, electrical conductivity, ammonia, nitrates+nitrites, total nitrogen and total phosphorus) and one biotic (chl-a) variable, and dimensionality reduced with principal component analysis (PCA). The component scores were used as independent variables in a multiple regression and the relationship between scores (indirect variables) and bacterial abundance was examined. An elevated bacterial abundance was observed during the limnophase (floodplain isolated from the river) and potamophase (flood). During the limnophase also the highest chl-a concentrations were found and in Sakadaš Lake during September 2003 significantly higher numbers of eutrophic than oligotrophic bacteria emerged (p = 0.026). During potamophase the opposite state was established. Elevated bacterial abundance occurred in parallel with minimal chl-a concentrations and during July 2004 in the Čonakut Channel a significantly higher number of oligotrophic than eutrophic bacteria emerged (p < 0.001). Differentiation in the number of eutrophic and oligotrophic bacteria suggests the existence of a higher concentration of labile DOC during September 2003 than during July 2004. Multiple regression analysis explains 13.6% of the variation in abundance of eutrophic bacteria. The regression model for oligotrophic bacteria is not significant. The results suggest two different mechanisms control bacterioplankton numbers to some extent in the Kopački Rit floodplain. In addition, two sources of DOC differentiating the quality of organic matter predominate under totally different hydrological regimes. Under these conditions, eutrophic bacteria may be partially bottom-up controlled, whereas it is not clear how the abundance of oligotrophs is controlled.  相似文献   

5.
In anaerobic coastal sediments, hydrolytic and/or fermentative bacteria degrade polymeric material and produce labile intermediates, which are used by terminal metabolizers to complete the conversion of organic material to CO2. We used molecular approaches to evaluate the response of two bacterial terminal metabolizer groups from a coastal tidal creek sediments, sulfate reducers and methanogens, to controlled changes in carbon resource supply. Tidal creek sediment bioreactors were established in April and August 2004. For each date, intact sediment sections were continuously supplied with flowthrough seawater that was either unamended or amended with the high-molecular-weight polysaccharide dextran. Biogeochemical data indicate that the activity of fermenting bacteria and the terminal metabolizers was limited by organic carbon supply during both experiments, with a significant increase in net volatile fatty acid (VFA) production and rates of sulfate reduction and methanogenesis following dextran addition. Community composition (measured by using terminal restriction fragment length polymorphism analysis, and functional gene [dsrA, mcrA] clone libraries) changed from April to August. However, community composition was not different between amended and unamended cores within each month, despite the change in resource level. Moreover, there was no relationship between community richness and evenness with resource level. This lack of variation in community composition with C addition could be attributed to the dynamic environment these sediment communities experience in situ. Fluctuations in VFA concentrations are most likely very high, so that the dominant bacterial species must be able to outcompete other species at both high and low resource levels.  相似文献   

6.
海水富营养化对海洋细菌影响的研究进展   总被引:2,自引:0,他引:2  
张瑜斌  章洁香  孙省利 《生态学报》2012,32(10):3225-3232
综述了海水富营养化对海洋细菌影响的研究进展。随着海水富营养化程度的增加,海洋细菌数量或生物量增加;反硝化细菌、大肠菌群尤其是厌氧性的硫酸盐还原菌和产甲烷菌等典型细菌生理群数量增加;浮游细菌群落结构随富营养化递增趋于简单,物种多样性降低;富营养化也明显导致细菌群落正常功能活性的紊乱。海水富营养化对细菌群落的结构和功能有着深远的影响。  相似文献   

7.
Microbial community structure in polluted Baltic Sea sediments   总被引:1,自引:0,他引:1  
Nearly half the seabed of the Baltic Proper is incapable of supporting life of higher organisms as a consequence of oxygen depletion resulting from eutrophication. However, these areas are actually teeming with microbial life. Here we used terminal-restriction fragment length polymorphism (T-RFLP) to investigate the dominant archaeal and bacterial groups, with respect to community structure, in surface layers of bottom sediments of the Baltic Sea along a coastal pollution gradient. Both archaeal and bacterial communities formed distinct clusters along the pollution gradient and the community compositions were different at the polluted sites compared with the relatively clean reference sites. The structures of the bacterial communities were most strongly correlated to water depth, followed by organic carbon, oxygen, salinity and silicate levels. In contrast, the structures of the archaeal communities were most strongly correlated to oxygen, salinity, organic carbon, silicate and nitrate levels. Some members of the microbial communities were identified using a combination of traditional and molecular approaches. Isolates obtained on different culture media were identified by partial sequencing of their 16S rRNA genes and some novel species were found. In addition, we developed a computer program, aplaus, to elucidate the putative identities of the most dominant community members by T-RFLP.  相似文献   

8.
Spatial and temporal variations in sediment microbial community structure in a eutrophic lake polluted with inorganic mercury were identified using polar lipid fatty acid (PLFA) analysis. Microbial community structure was strongly related to mercury methylation potential, sediment organic carbon content, and lake location. Pore water sulfate, total mercury concentrations, and organic matter C/N ratios showed no relationships with microbial community structure. Seasonal changes and changes potentially attributable to temperature regulation of bacterial membranes were detectable but were less important influences on sediment PLFA composition than were differences due to lake sampling location. Analysis of biomarker PLFAs characteristic of Desulfobacter and Desulfovibrio groups of sulfate-reducing bacteria suggests that Desulfobacter-like organisms are important mercury methylators in the sediments, especially in the Lower Arm of Clear Lake.  相似文献   

9.
In Eastern Harbour (EH) of Alexandria (Egypt), where an under-water museum is planned to be built, the distribution of some heterotrophic bacteria and their relationships to physical parameters and biochemical composition of the sedimentary organ ic matter were investigated. The Eastern Harbour is a relatively shallow semi-closed basin and is sheltered from the sea by an art ificial breakwater leaving two openings, El-Boughaz and El-Silsila. Seven stations covering the area of the EH were selected and surface sediments were collected seasonally from spring to winter 2002. The near bottom temperature varied seasonally with a minimum value in winter and a maximum in summer. In contrast to the temperature values, dissolved oxygen exhibited maximum values in cold seasons. The seasonal average of the total organic carbon ranged from 0.48 ± 0.16 to 4.42 ± 2.46%, while the total organic nitroge n ranged from 0.07±0.08 to 0.42±0.38%. The total carbohydrate had minimum and maximum values of 273 μg g?1 and 6539 μg g?1. The combined amino acids represented the dominant biochemical class of organic matter in the EH sediments with an average of 365 ± 1911 μg g?1. The total bacterial count ranged from 1.4 × 104 to 1.4 × 107 colony forming unit (CFU) g?1 sediment dry weight. Amylolytic bacterial group was recorded in almost all sites and seasons, while proteolytic bacteria were dominant in spring and au tumn. The variation in the abundance of amylolytic and proteolytic bacterial groups was found to be parallel to the variation in soluble carbohydrates and free amino acids. High percentage of H2S-producing bacteria was reported during summer at some stations confirming the low oxygen content of the sediment at these sites. Agar-degrading bacteria were found only in warm seasons. The count of co liform bacteria in the EH sediments was very low (<10 CFU g?1) during all monitored seasons indicating that the EH marine environment was almost free of domestic waste discharge during this period.  相似文献   

10.
The deep sea is a unique and extreme environment characterized by low concentrations of highly recalcitrant carbon. As a consequence, large organic inputs have potential to cause significant perturbation. To assess the impact of organic enrichment on deep sea microbial communities, we investigated bacterial diversity in sediments underlying two whale falls at 1820 and 2893 m depth in Monterey Canyon, as compared with surrounding reference sediment 10–20 m away. Bacteroidetes, Epsilonproteobacteria and Firmicutes were recovered primarily from whale fall‐associated sediments, while Gammaproteobacteria and Planctomycetes were found primarily within reference sediments. Abundant Deltaproteobacteria were recovered from both sediment types, but the Desulfobacteraceae and Desulfobulbaceae families were observed primarily beneath the whale falls. UniFrac analysis revealed that bacterial communities from the two whale falls (~30 km apart) clustered to the exclusion of corresponding reference sediment communities, suggesting that deposition of whale fall biomass is more influential on deep sea microbial communities than specific seafloor location. The bacterial population at whale‐1820 at 7 months post deposition was less diverse than reference sediments, with Delta‐ and Epsilonproteobacteria and Bacteroidetes making up 89% of the community. At 70 months, bacterial diversity in reference sediments near whale‐2893 had decreased as well. Over this time, there was a convergence of each community's membership at the phyla level, although lower‐taxonomic‐level composition remained distinct. Long‐term impact of organic carbon loading from the whale falls was also evident by elevated total organic carbon and enhanced proteolytic activity for at least 17–70 months. The response of the sedimentary microbial community to large pulses of organic carbon is complex, likely affected by increased animal bioturbation, and may be sustained over time periods that span years to perhaps even decades.  相似文献   

11.
Knowledge of the bacterial community structure in sediments is essential to better design restoration strategies for eutrophied lakes. In this regard, the aim of this study was to quantify the abundance and activity of bacteria involved in nutrient and iron cycling in sediments from four Azorean lakes with distinct trophic states (Verde, Azul, Furnas and Fogo). Inferred from quantitative PCR, bacteria performing anaerobic ammonia oxidation were the most abundant in the eutrophic lakes Verde, Azul and Furnas (4.5-16.6%), followed by nitrifying bacteria (0.8-13.0%), denitrifying bacteria (DNB) (0.5-6.8%), iron-reducing bacteria (0.2-1.4%) and phosphorus-accumulating organisms (<0.3%). In contrast, DNB dominated sediments from the oligo-mesotrophic lake Fogo (8.8%). Activity assays suggested that bacteria performing ammonia oxidation (aerobic and anaerobic), nitrite oxidation, heterothrophic nitrate reduction, iron reduction and biological phosphorus storage/release were present and active in all Azorean lake sediments. The present work also suggested that the activity of DNB might contribute to the release of phosphorus from sediments.  相似文献   

12.
【目的】城市湖泊沉积物中的微生物群体是富营养环境生物修复的重要因素。本研究拟对中型富营养化湖泊——武汉东湖沉积物细菌群体结构及空间变化规律进行研究。加深对富营养湖泊微生物群体结构的理解,并为城市湖泊的保护和污染控制提供参考。【方法】采用16S r RNA基因RFLP分析和序列分析技术获得细菌群体系统发育信息。采用PCoA法分析武汉东湖沉积物细菌群体内以及与其它湖泊沉积物细菌群体间的相似性。采用CCA法分析环境因素对细菌群体的影响。【结果】武汉东湖沉积物细菌群体包含13个门和2个未知种群。PCoA分析进一步表明来自东湖3个子湖的细菌群体与其它地域富营养化湖泊的微生物种群结构相似,并区别于重度富营养化子湖庙湖的细菌群体。在庙湖中,我们发现了Thermogymnomonas类古菌的含量显著偏高。CCA分析表明细菌的分布与沉积物中碳、氮和磷元素的含量显著相关。【结论】本研究进一步确证了环境因素对细菌群体组成和分布的调节作用,加深了对富营养水体微生物群体结构的理解,并为城市湖泊的保护和污染控制提供了参考。  相似文献   

13.
Despite of the importance for tracking sources and fates of organics in determining carbon cycling and assessing the overall health of lacustrine ecosystems, little is known of organics behaviors in shallow eutrophic lakes. In this study, for relating the benthic organics to pelagic sources, an ecosystem-level investigation was performed in hypereutrophic Lake Taihu, China, by using source-specific fatty acid biomarkers on the molecular level. Results exhibited the unexpected asymmetric phenomena of organics between surface water and sediments. In the abundance, the dominant organics shifted from cyanobacteria in surface water to terrestrial plants in sediments. In the spatial pattern, as opposed to terrestrial plants, cyanobacteria were found not site-to-site symmetrical, although both of their spatial distributions varied strongly. Essentially, these asymmetric phenomena ascribed to a more considerable loss of cyanobacteria compared to terrestrial plants during sinking, caused by aquatic animal consumption and microbial utilization with different degree. Combined dual stable isotopes (δ13C and δ15N) and fatty acid biomarkers revealed that there were only subtle differences in the diets of benthic and pelagic animals and cyanobacteria were their main food source. Concomitantly, results of δ13C of bacteria-specific fatty acids demonstrated that bacteria equally and profoundly affected organics accumulation or preservation in the sediments, because they preferentially utilized labile cyanobacteria as their carbon source instead of terrestrial plants (>95% within these two sources). Consequently, these novel findings clarify that not only in deep lakes, but also shallow eutrophic ones, the extensive losses of autochthonous organic matter can be expected during sedimentation coupling with the dramatical modifications of biogeochemical processes.  相似文献   

14.
15.
Abstract. The gutless marine oligochaete, Olavius crassitunicatus finogenova 1986 (Tubificidae), from suboxic to sulfidic sediments off Peru, consistently harbored 3 structurally distinct types of extracellular bacterial symbionts. Large, oval bacteria were labeled immunocytochemically, proving their nature as autotrophs. Spectroscopical analysis documented storage of sulfur in this numerically dominant morphotype. Small, rod-shaped bacteria attained a more peripheral position adjacent to the cuticle. The third bacterial type was represented by long, filamentous forms which were often in close contact to the oval bacteria. With their curved or undulate cells, these filiform bacteria resembled spirochetes. They were clearly distinguishable and consistently found in all investigated host specimens.
While molecular analyses could not be performed, structural and immunocytochemical evidence indicated that the oval bacteria seemed equivalent to the γ-proteobacteria in related gutless oligochaetes. On the basis of morphological similarity and indications from closely related symbiotic tubificids, the possible relationship of the two other morphoptypes must remain unsolved and needs further molecular analysis. The three bacterial morphotypes live in a consistent, elaborate, and apparently obligate coexistence with a host that has completely reduced its digestive and excretory organs.  相似文献   

16.
Dziuban AN 《Mikrobiologiia》2005,74(1):119-125
The cell numbers and ecological characteristics of the distribution of certain species of butyric acid bacteria (BAB) of the genus Clostridium in the bottom sediments of inland basins of different types were studied using the optimal nutrient media. The seasonal dynamics of clostridial vegetative cells and spores in sediments with different ecological conditions were revealed. The cell numbers of the dominant BAB species were shown to depend on the redox potential of the sediments, the amount and composition of C(org), and the trophic state of the basin in general. C. pasteurianum was found to predominate in eutrophic lakes and reservoirs (5-11 x 10(6) cells/cm3), C. butyricum and C. felsineum predominated in mesotrophic ones (2-11 x 10(6) cells/cm3), and C. acetobutylicum was predominant in acidic chthionoeutrophic lakes and reservoirs (0.1-0.5 x 10(6) cells/cm3). The lowest cell numbers of BAB were found in river sediments, whereas the highest numbers were recorded in the sediments of polysaprobic zones (0.1-1.0 x 10(3) and 0.5-2.0 x 10(7) cells/cm3 respectively).  相似文献   

17.
黄河内蒙古段表层沉积物细菌多样性及群落结构类型   总被引:5,自引:0,他引:5  
为掌握黄河内蒙古段表层沉积物微生物多样性、群落结构类型及其影响因素,采用高通量测序技术分析了6个采样点表层沉积物中的微生物多样性及群落结构。研究结果表明,黄河内蒙古段沉积物细菌丰度大小排序为乌拉特前旗(H3)老牛湾(H6)临河(H2)包头(H4)托县(H5)乌海(H1),微生物多样性排序为H6 H2 H5 H4 H3 H1,乌海沉积物中细菌丰度和微生物多样性都是最低的一个采样点。黄河内蒙古段表层沉积物中三大优势菌群分别为变形菌门(Proteobacteria,相对丰度32.39%)、绿弯菌门(Chloroflexi,13.25%)和拟杆菌门(Bacteroidetes,12.16%)。细菌群落丰度与环境因子之间的冗余分析结果显示,沉积物中总有机碳(TOC)、离子交换容量(CEC)和总磷(TP)、总氮(TN)等环境因子对黄河内蒙古段沉积物细菌群落分布影响较大,负相关系数分别为82.5%、80.1%、85.5%和85.2%;微生物多样性与环境理化因子相关性分析结果表明,黄河沉积物微生物多样性格局与对氮磷等营养物质的损耗有直接关系。  相似文献   

18.
Hu B  Shen L  Du P  Zheng P  Xu X  Zeng J 《PloS one》2012,7(3):e33826
Continuous chemical pollution is one of the most serious environmental problems in the Jiaojiang Estuary of the East Sea (China). This chemical pollution has significantly changed the estuarine environmental conditions and may have profoundly influenced the distribution of anammox bacterial communities in this estuary. Here, we investigated the influence of chemical pollution on the community composition, diversity and abundance of anammox bacteria in Jiaojiang estuarine sediments. Phylogenetic analysis of 16S rRNA genes showed that the majority of anammox bacterial sequences retrieved from the estuarine intertidal sediments were associated with Kuenenia. In contrast, different anammox communities composed of Brocadia, Kuenenia, Scalindua and Jettenia were found in the estuarine subtidal sediments. Redundancy analysis (RDA) indicated that the sediment nitrobenzene and organic content had significant impacts on the distribution of anammox communities in the intertidal sediments. Pearson correlation analysis showed that the diversity of anammox bacteria in the intertidal sediments was positively correlated with the organic content. In contrast, RDA results showed that the nitrobenzene content, NO(3)(-) concentration and salinity significantly influenced the distribution of anammox communities in the subtidal sediments. The diversity and relative abundance of anammox bacteria in the subtidal sediments were positively correlated with NO(3)(-) concentration.  相似文献   

19.
The impact of a sewage point source on the bacterial densities in an intertidal mud flat in Boston Harbor, Mass., was investigated. The area, Savin Hill Cove, acts as a receiving basin for a combined storm and sewage outlet (CSO). Preliminary examination of sediments and overlying water at high tide demonstrated that fecal coliforms were present in sediments at abundances 2 to 4 orders of magnitude higher than in the overlying water column. The following bacterial counts were determined from sediments along a sampling transect extending 460 m from the CSO: total bacteria by epifluorescent microscopy, heterotrophic bacteria by plate counts on nutrient-rich and nutrient-poor media, fecal coliforms and enterococci by membrane filtration, and Vibrio parahaemolyticus by a most-probable-number technique with a resuscitation step. Median sediment grain size, average tidal exposure, carbon/nitrogen ratio, and total organic carbon were also measured. All bacterial indices, except for V. parahaemolyticus, declined significantly with distance from the outfall. Multiple regression analysis indicated that tidal exposure (low tides) may affect densities of total bacteria. Fecal coliforms and enterococci were still present in appreciable numbers in sediments as far as 460 m away from the CSO. In contrast, V. parahaemolyticus densities did not correlate with the other bacterial counts nor with any of the environmental parameters examined. These results indicate that intertidal sediments which adjoin point sources of pollution are severely contaminated and should be considered as potentially hazardous reservoirs of sewage-borne diseases.  相似文献   

20.
Top-down control of lower trophic levels, e.g., bacteria, has been suggested to increase along aquatic productivity gradients. The response by the bacterial community may be to become more predation resistant in highly productive environments. To test this hypothesis, samples were taken from 20 aquatic systems along a productivity gradient (dissolved organic carbon from 7 to 71 mg/L), during late summer. The results showed that the biomass of bacteria, phytoplankton, and ciliates increased along the gradient (r2 = 0.532, 0.426, and 0.758, P < 0.01, respectively). However, the organisms did not increase equally, and the ratio of protozoan to bacterial biomass showed a 100-fold increase along the gradient. Ciliates dominated the protozoan biomass in the more nutrient-rich waters. The edibility of colony-forming bacteria was tested using a ciliate predator, Tetrahymena pyriformis. Bacterial edibility was found to decrease with increases in nutrient richness and ciliate biomass in the aquatic systems (r2 = 0.358, P < 0.01; r2 = 0.242, P < 0.05, respectively). Quantile regression analysis indicated that the selection pressures on edible bacteria were increasing along the productivity gradient. Thus, inedible forms of bacteria were selected for in the transition from oligotrophic to eutrophic conditions. Isolated bacteria were distributed among the alpha-, beta-, and gamma- Proteobacteria and the Actinobacteria and Firmicutes taxa. We conclude that bacterial predation resistance increases in nutrient-rich waters with high protozoan predation.  相似文献   

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