Use of an Exotic Carbon Source To Selectively Increase Metabolic Activity and Growth of Pseudomonas putida in Soil

Respiration and growth of Pseudomonas putida PpG7, containing catabolic plasmid NAH7, was determined in three agricultural field soils amended with the carbon source salicylate. The addition of salicylate to soil significantly increased the population of PpG7. However, there was a lack of relationship between microbial numbers and activity as determined by evolution of CO₂. In soils containing 30 to 1,500 μg of salicylate per g, metabolic activities of PpG7 peaked between 18 and 42 h and population densities increased approximately 10¹-to 10⁵-fold. However, the metabolic activity of PpG7 rapidly declined after salicylate was utilized, whereas peak population densities were maintained for the duration of the experiments (5 to 7 days). Thus, elevated population densities of PpG7 were represented by inactive cells. Soil type had only minor effects on respiration rates or growth curves of PpG7 when amended with comparable concentrations of salicylate. Respiration and growth rates were optimal at concentrations between 300 and 1,000 μg of salicylate per g in the test soils. At 1,500 to 2,500 μg/g, respiration and growth of PpG7 were initially suppressed, but after a short lag time both attained levels similar to or greater than those resulting from the use of lower concentrations of salicylate. The culturing of PpG7 on a salicylate-amended medium to induce salicylate-degradative enzymes did not affect the lag time before utilization of salicylate in soil. Although PpG7 competed well with fungi for the substrate, suppression of fungal populations with cycloheximide resulted in significantly increased population densities of PpG7 in two of three soils amended with salicylate. The beneficial activities of bacteria in soil are discussed in relation to population density, population metabolic activity, and selective carbon source utilization.     

土壤中镉对丛枝菌根真菌Glomus mosseae生长的效应

采用分室培养法,在同一宿主植物生长的土壤中设含有0、5、25、50mg/kg4个不同浓度重金属镉的菌丝生长室(用30μm尼龙网与根系隔开)以期建立在宿主植物生长状况完全相同的条件下研究环境因素对AM真菌直接影响的新方法,并在此基础上探讨不同浓度的重金属镉对丛枝菌根真菌Glomus mosseae(BEG167)生长的直接影响。结果表明,与不施加镉的处理相比,土壤中施加低浓度镉(5mg/kg)刺激了G.mosseae的生长,其菌丝总长度最大;高浓度镉(大于25mg/kg)抑制了G.mosseae的生长,其菌丝总长度较小。AM真菌的代谢活性与土壤镉浓度的关系也表现出与菌丝生物量相同的规律。以上结果表明:G.mosseae在镉污染环境中有应激反应的特征,即:当G.mosseae受到轻微毒害时,为了适应其生存条件的改变而不断增加其代谢活性和生长量来降低镉的毒害。此外,本方法用于研究宿主植物生长状况相同的条件下,重金属毒害或其他环境因素对AM真菌生长代谢的直接影响是可行的。     

A novel catabolic activity of Pseudomonas veronii in biotransformation of pentachlorophenol

Pseudomonas veronii PH-05, a bacterial strain capable of transforming pentachlorophenol (PCP) to a metabolic intermediate, was isolated by selective enrichment of soil samples from a timber storage yard. Strain PH-05 was shown to be able to grow using PCP as the sole source of carbon and energy. GC-MS analysis showed that the metabolic intermediate was tetrachlorocatechol, which inhibited the growth of this strain. The formation of tetrachlorocatechol during biotransformation was monitored, and its inhibitory effect on growth of strain PH-05 was analyzed at a range of concentrations. The catabolic activity of the isolated strain differs from that of other PCP-degrading bacteria, which metabolize PCP through a chlorinated hydroquinone intermediate.     

Effect of the insecticide nerametrine EK-15 on the activity of soil microorganisms

The effect of the insecticide Nerametrine EK-15 (containing an active supercypermetrine component) on nitrification, nitrogen fixation, CO₂ production and cellulase activity of soil microorganisms was investigated. Four soil types were sampled from various localities. Supercypermetrine at 31 pmol/kg soil affected remarkably the metabolic activity of all soil samples tested by producing CO₂ after a 1-d exposure. After a 14-d exposure no difference in the metabolic activity related to CO₂ production was noticed in the case of garden soil where the insecticide at 31 pmol/kg soil and the unaffected control were used. As far as other samples are concerned, the supercypermetrine concentration amounting to 31 pmol/kg soil explicitly inhibited the metabolic activity of soil microorganisms. On the other hand, concentrations of 0.61 and 6.1 pmol/kg soil stimulated the metabolic activity of soil in the locality of Senica. The soil samples enriched with nutrients (organic nitrogen in urea) manifested an evident inhibition at 31 pmol/kg soil. The nitrification activity of all soil types was interrupted at 61 pmol/kg soil. Supercypermetrine 0.12 pmol/L stopped completely nitrogen fixation withA. chroococcum and that corresponding to 0.3 pmol/L stopped aerobic cellulase decomposition.     

土壤微生物总活性研究方法进展

微生物总活性是指在某一时段内微生物所有生命活动的总和,它直接决定着微生物行使生理、生态功能的能力,是微生物学研究的热点,也是难点。迄今为止,还没有建立直接测定微生物总活性的方法,只能用一些相关指标来间接反映它。目前常用的指标主要包括微生物的呼吸速率、生长速率以及胞内RNA含量等。与其它一些基质和环境相比,测定土壤中的微生物总活性更为困难。通过总结研究土壤微生物总活性常用的3种方法,在简略概括传统的土壤微生物呼吸测定法的基础上,详细介绍了放射性同位素标记法和RNA直接表征法的原理和操作流程,整理归纳了一些重要应用案例,比较分析了不同方法的优缺点,以期为选择研究土壤微生物总活性的适宜方法提供依据。     

Changes in soil CO2 and O2 concentrations when radiata pine is grown in competition with pasture or weeds and possible feedbacks with radiata pine root growth and respiration

This study examined the reciprocal effects of growing ryegrass, lotus and other weed species in competition with radiata pine on soil CO₂ and O₂ concentrations and on the growth and root respiration of the radiata pine. Soil O₂ concentrations decreased and soil CO₂ concentrations increased with increasing soil depth. Radiata pine plus competing species slightly reduced soil O₂ concentrations and markedly increased soil CO₂ concentrations (up to 40 mmol mol⁻¹) compared with radiata pine alone. The dry weights of shoots and roots, and the root respiration rates of radiata pine grown with competing vegetation were much less than those for radiata pine alone. This probably was not solely caused by competition for nutrients water or light since adequate water and nutrients were supplied to all treatments and the radiata pine overtopped the competing vegetation. When radiata pine roots were raised in NaHCO₃ solutions equivalent to a range of CO₂ concentrations, succinate dehydrogenase activity (a metabolic indicator of mitochondrial respiration) and elongation rates of roots decreased as CO₂ concentrations increased from 0 to 40 mmol mol⁻¹. This suggests that the elevated CO₂ concentrations found in the experiments in soil was the cause, at least in part, of the reduced growth of radiata pine in competition with other species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Population Changes of Soil Microbial Communities Induced by Hydrocarbon and Heavy Metal Contamination

Substrate utilization tests with Biolog® plates were used to obtain information on shifts in community composition and on changes in the metabolic diversity and activity of microorganisms in soil polluted with hydrocarbons. and/or heavy metals. Differences between the patterns of substrate utilization of endogenous microorganisms of pristine and contaminated soils were investigated by multivariate analysis. Population changes and shifts in metabolic diversity were observed both after hydrocarbon pollution or heavy metal contamination. The overall activity on the 95 Biolog® Gram-negative (GN) substrates correlated well with the respiration rate of the soil. Soils contaminated with hydrocarbons showed higher metabolic potentials than the corresponding controls. In contrast, heavy metal pollution caused both lower metabolic activity and a loss in diversity. The Biolog® assay was found to be suitable to describe changes in functional diversity of soils caused by hydrocarbon contamination or heavy metal stress.     

玉米幼苗根际土壤微生物活性对芘污染的响应

用根际袋法土培试验研究了玉米幼苗根际与非根际土壤微生物量碳、微生物熵、代谢熵和土壤酶活性对不同芘污染水平(50、200、800mg·kg-1,记为T1、T2、T3)的响应差异。结果表明,较低浓度芘可适当的刺激玉米幼苗的生长,而较高浓度芘则抑制幼苗生长,其抑制作用随芘处理浓度的提高而增强;芘对玉米根系的影响要大于对茎叶的影响。玉米幼苗能够明显促进土壤中芘的去除。根际和非根际土壤中芘的去除率分别为56.67%-76.18%和32.64%-70.44%,根际土壤中芘的平均去除率比非根际土壤高16.06%。同处理中根际土壤芘含量显著低于非根际土壤,随着芘处理浓度的提高其差异更加显著。根际土壤微生物量碳、微生物熵、多酚氧化酶活性、脱氢酶活性和磷酸酶活性均高于非根际土壤,代谢熵低于非根际土壤,且其差异随芘处理浓度的提高而增大。在不同芘污染水平下,微生物量碳、微生物熵和脱氢酶活性根际和非根际土壤为T1T2T3,代谢熵为T3T2T1;多酚氧化酶活性根际土壤为T2T1T3,非根际土壤为T1T2T3;磷酸酶活性根际土壤为T3T1T2,非根际土壤为T1T2T3。土壤中残余芘含量与土壤微生物量碳、微生物熵、多酚氧化酶、脱氢酶和磷酸酶活性呈显著负相关,与代谢熵呈显著正相关。     

Determination of metabolic activity of streptomycetes in soil microcosms

Two Streptomyces griseus strains were isolated from different soil types. S. griseus CAG17 strain was isolated from an agricultural area with low organic matter but rich in phosphorus content and S. griseus 26K strain was isolated from a forest area rich in organic matter with a low phosphorus content. The survival and metabolic activity of these isolates were studied in dynamic sterile soil microcosm systems. The fitness of each isolate was studied by re-inoculation in a soil type different from its origin. Maximum percentage of germination and respiration rates occurred within the first 48 h after each soil turnover (removal and addition of certain soil volumes). Data suggested that S. griseus CAG17 survived better independently of the soil type in comparison with S. griseus 26K which sporulated within the first 12 h after inoculation. Incubation temperatures did affect the lifecycles in relation to soil type. For example, the lowest temperature tested, 22 degrees C, was more favourable for extended germination and adaptation in general but revealed lesser spore numbers in the 'foreign' soil environment. Monitoring metabolic activity by estimation of urease, phosphatases and dehydrogenase-specific activities, between 18 and 35 degrees C incubation temperatures, was a reliable method for studying the survival and growth of streptomycete populations in soil. Results also confirmed that respiration rate and enzyme-specific activity corresponded with spore counts in long-term experiments which were designed for the investigation of survival and growth of S. griseus CAG17. Under selective pressure by heavy metals, in soil microcosm systems, metabolic activity proved a useful tool for the investigation of streptomycete activity. These methods could also be applied in agricultural field studies for monitoring microbial populations under conditions where various 'pollutants' are present in soil samples.     

Use of an isothermal microcalorimetry assay to characterize microbial oxalotrophic activity

Isothermal microcalorimetry (IMC) has been used in the past to monitor metabolic activities in living systems. A few studies have used it on ecological research. In this study, IMC was used to monitor oxalotrophic activity, a widespread bacterial metabolism found in the environment, and particularly in soils. Six model strains were inoculated in solid angle media with K-oxalate as the sole carbon source. Cupriavidus oxalaticus, Cupriavidus necator, and Streptomyces violaceoruber presented the highest activity (91, 40, and 55 μW, respectively) and a maximum growth rate (μmax h(-1) ) of 0.264, 0.185, and 0.199, respectively, among the strains tested. These three strains were selected to test the incidence of different oxalate sources (Ca, Cu, and Fe-oxalate salts) in the metabolic activity. The highest activity was obtained in Ca-oxalate for C. oxalaticus. Similar experiments were carried out with a model soil to test whether this approach can be used to measure oxalotrophic activity in field samples. Although measuring oxalotrophic activity in a soil was challenging, there was a clear effect of the amendment with oxalate on the metabolic activity measured in soil. The correlation between heat flow and growth suggests that IMC analysis is a powerful method to monitor bacterial oxalotrophic activity.     

施用生物质炭和羊粪对宿根连作茶园根际土壤微生物的影响

茶树长期宿根连作会导致土壤酸化严重、土壤营养不平衡、根际土壤微生态结构恶化.研究生物质炭、羊粪对宿根连作茶树生长以及土壤微生物群落结构和功能的影响,探讨其对宿根连作茶树土壤环境的调节效果,可为宿根连作茶园土壤微生态的改善提供理论依据.本研究以宿根连作20年的茶园土壤为对象,利用Biolog技术和磷脂脂肪酸(PLFA)方法,研究施用生物质炭(40 t·hm^-2)和羊粪替代部分化肥对连作茶树产量和品质、土壤化学性质、根际土壤微生物群落功能和结构的影响.结果表明:施用生物质炭、羊粪1年后酸化茶园土壤的p H和土壤养分显著提高,并提高了茶叶产量.与常规施肥相比,施用生物质炭、羊粪替代部分化肥处理显著提高了茶树根际土壤微生物的碳源代谢活性和微生物多样性,对胺类、碳水化合物和聚合物的相对利用有所增加.生物质炭和羊粪处理的根际土壤总PLFA含量分别比常规施肥处理提高了20.9%和47.5%,羊粪处理还显著降低了总饱和/总单一不饱和脂肪酸比例.生物质炭和羊粪可改善茶园土壤酸化状况和土壤肥力,对宿根连作茶树的生长具有促进作用,两种措施均不同程度地增加了土壤微生物的代谢活性和微生物量、提高了多样性指数、改善了微生物群落结构.施用生物质炭和羊粪可作为调节宿根连作茶园根际土壤微生态的有效措施.     

Impact of an Herbicide Combination of Bromoxynil and Prosulfuron on Soil Microorganisms

Soil microcosm experiments were used to investigate the effects on growth and activity of soil microorganisms of an herbicide combination (60% bromoxynil + 3% prosulfuron) frequently used to provide a broad spectrum control of weed species. Culturable aerobic bacteria, fungi, and actinomycetes, the fundamental groups of heterotrophic microorganisms, and nitrifiers, considered a very sensitive group to these compounds, were evaluated. Since herbicides have been found to inhibit decomposition of cellulose in soil, the effects on cellulolytic bacteria and fungi were determined. Dehydrogenase activity as a measure of microbial activity was another parameter considered. The results emphasized a tendency of reversible stimulatory/inhibitory effects of the tested compounds on soil microorganisms, with fungi as an exception. A long-lasting negative action on the activity of the dehydrogenase (DHA), commonly used as an index of the overall microbial activity in soil, was found. The magnitude of these effects were dependent on the assayed concentrations of the herbicides mixture. We concluded that the presence of bromoxynil + prosulfuron could induce significant changes in the microbial populations of the soil, concerning the activity and balance of microbial community. Possible environmental risks must be considered. Dehydrogenase activity was shown to be an important indicator of side-effects attributed to these herbicides.     

有机磷杀虫剂——灭克磷对丛枝菌根真菌Glomus mosseae生长的效应

采用多室隔网培养法,在宿主植物生长状况相同的条件下研究了土壤中不同浓度灭克磷对AM真菌生长的直接效应。与对照相比,0.5、1.5和3.5mg/kg灭克磷使Glomusmosseae菌丝生长量和具有琥珀酸脱氢酶活性的菌丝生长量明显地增加;随着灭克磷剂量的增加,菌丝总量的相对生长速率呈降低趋势,但在数值上仍然高于对照的;与此同时,高剂量(1.5和3.5mg/kg)灭克磷使Glomusmosseae活性菌丝量的相对增长速率明显低于对照,达到几乎停止的水平。上述结果说明,低剂量灭克磷(0.5mg/kg)对AM真菌生长和代谢活性都有一定刺激作用,但是在高剂量灭克磷(1.5和3.5mg/kg)下菌丝生物量的增加和周转速度的加快只是AM真菌对毒物的应激反应。     

Metabolomics Suggests That Soil Inoculation with Arbuscular Mycorrhizal Fungi Decreased Free Amino Acid Content in Roots of Durum Wheat Grown under N-Limited,P-Rich Field Conditions

Arbuscular mycorrhizal fungi (AMF) have a major impact on plant nutrition, defence against pathogens, a plant’s reaction to stressful environments, soil fertility, and a plant’s relationship with other microorganisms. Such effects imply a broad reprogramming of the plant’s metabolic activity. However, little information is available regarding the role of AMF and their relation to other soil plant growth—promoting microorganisms in the plant metabolome, especially under realistic field conditions. In the present experiment, we evaluated the effects of inoculation with AMF, either alone or in combination with plant growth–promoting rhizobacteria (PGPR), on the metabolome and changes in metabolic pathways in the roots of durum wheat (Triticum durum Desf.) grown under N-limited agronomic conditions in a P-rich environment. These two treatments were compared to infection by the natural AMF population (NAT). Soil inoculation with AMF almost doubled wheat root colonization by AMF and decreased the root concentrations of most compounds in all metabolic pathways, especially amino acids (AA) and saturated fatty acids, whereas inoculation with AMF+PGPR increased the concentrations of such compounds compared to inoculation with AMF alone. Enrichment metabolomics analyses showed that AA metabolic pathways were mostly changed by the treatments, with reduced amination activity in roots most likely due to a shift from the biosynthesis of common AA to γ-amino butyric acid. The root metabolome differed between AMF and NAT but not AMF+PGPR and AMF or NAT. Because the PGPR used were potent mineralisers, and AMF can retain most nitrogen (N) taken as organic compounds for their own growth, it is likely that this result was due to an increased concentration of mineral N in soil inoculated with AMF+PGPR compared to AMF alone.     

Microbial metabolic activity in soil as measured by dehydrogenase determinations.

The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 degrees C incubation with either glucose of yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.     

Microbial metabolic activity in soil as measured by dehydrogenase determinations.

The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 degrees C incubation with either glucose of yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.     

An increase in the immune system activity of the wax moth Galleria mellonella and of the Colorado potato beetle Leptinotarsa decemlineata under effect of organophosphorus insecticide

Several aspects of immune response in insects treated with an organophosphate (pirimiphosmethyl) have been assessed. Both humoral (phenoloxidase activity) and cellular (hemocyte count and encapsulation rate) immunity changes have been studied in larvae of the representatives of two insect orders, the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera, Chrysomelidae) and the wax moth Galleria mellonella (Lepidoptera, Pyralidae). A direct contact with sublethal and half-lethal doses of the insecticide results in stimulation of immune reactions: phenoloxidase activity and encapsulation rate are heightened, and hemocyte count increases.     

Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere.

The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium. Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere. Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions. The DF57-P2 reporter strain responded to phosphate limitation, and the luxAB gene fusion was controlled by a promoter containing regulatory sequences characteristic of members of the phosphate (Pho) regulon. DF57 generally had higher metabolic activity in a gnotobiotic rhizosphere than in the corresponding bulk soil. Within the rhizosphere the distribution of metabolic activity along the root differed between the rhizosphere soil and the rhizoplane, suggesting that growth conditions may differ between these two habitats. The DF57-P2 reporter strain encountered phosphate limitation in a gnotobiotic rhizosphere but not in a natural rhizosphere. This difference in phosphate availability seemed to be due to the indigenous microbial population, as DF57-P2 did not report phosphate limitation when established in the rhizosphere of plants in sterilized soil amended with indigenous microorganisms.     

湿地土壤微生物功能多样性及碳氮组分对长期氮输入的响应

氮输入对湿地生态系统碳氮循环具有重要影响,研究湿地土壤微生物功能多样性及碳氮组分对氮输入的响应,对于明确湿地土壤碳氮循环微生物驱动机制具有重要意义。依托长期野外氮输入模拟试验,利用Biolog-ECO微平板技术,分析不同浓度氮输入:N1(6 g N m^-2 a^-1)、N2(12 g N m^-2 a^-1)和N3(24 g N m^-2 a^-1)对湿地土壤表层(0-15 cm)和亚表层(15-30 cm)微生物碳源代谢活性、功能多样性和碳氮组分的影响。结果表明:N2处理显著提高了亚表层土壤微生物碳源代谢活性和McIntosh指数,N3处理显著降低了表层土壤微生物Shannon指数和Shannon-evenness指数。随氮输入浓度增加湿地表层土壤微生物对糖类的利用率显著降低,N3处理表层土壤微生物对胺类的利用率以及亚表层土壤微生物对醇类的利用率显著提高。N1处理显著提高了湿地表层土壤全氮和微生物量碳含量;N2、N3处理显著提高了土壤铵态氮、硝态氮含量;N3处理显著降低了土壤pH值。湿地土壤pH、总碳、溶解性有机碳含量是影响微生物碳源代谢活性和功能多样性的重要因素,土壤溶解性有机碳、铵态氮、全氮含量、含水率是影响微生物碳源利用变化的主要因子。     

Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms

The growth and activity of introduced (S. lividans TK24 pIJ673 and S.lividans TK23) and indigenous (S.griseus CAG17) streptomycete strains in soil was studied, under controlled conditions. The effect of environmental parameters such as temperature, soil water content and nutrient availability on the growth and activity of these strains, was studied using a highly dynamic fed-batch soil microcosm system. Using this new system, repeated cycles of active streptomycete growth were achieved, allowing long-term investigation of metabolic activity, plasmid stability and conjugative plasmid transfer. In long-term experiments, respiration rates and enzyme activity patterns matched the pattern of germination/sporulation cycles of the inoculants. In situ hybridisation, using fluorescently labelled oligonucleotides, also proved the presence of metabolically active streptomycete mycelia in sterile soil. Plasmid stability under varying temperatures and selective pressure was studied using the above system. In both sterile and non sterile amended antibiotic containing soil, no intraspecific transfer of plasmid pIJ673 from S.lividans TK24 to S.griseus CAG17 was detected. The soil microcosm system used, though, permitted detection of intraspecific conjugative transfer of this plasmid from S.lividans TK24 to S.lividans TK23 in soil.