Periphytic Photosynthetic Stimulation of Extracellular Enzyme Activity in Aquatic Microbial Communities Associated with Decaying Typha Litter

We examined the effect of light on extracellular enzyme activities of periphytic/endogenous microbial assemblages associated with decomposing litter of an emergent macrophyte Typha angustifolia within a small inland wetland in southeastern Michigan. Standing-dead Typha leaf litter was collected, placed into floating wire mesh litter baskets, and submerged in a wetland pool. Enzyme saturation assays were conducted on three occasions following litter submergence (days 9, 28, and 44) to generate saturation curves for the individual enzymes tested and to examine potential differences in enzyme saturation kinetics during microbial colonization and development. Experimental light manipulations were conducted on two occasions during microbial development (days 10 and 29). Short-term (30 min) light exposure significantly increased extracellular β-glucosidase activity of litter-associated microbial communities. Activities of β-xylosidase and leucine-aminopeptidase were not stimulated, and stimulation of phosphatase activity was variable. The exact mechanism for increased enzyme activity remains unknown, but it may have been increased pH arising from periphytic algal photosynthesis. These results suggest that extracellular enzyme activity in microbial communities colonizing natural organic substrata may be influenced by light/photosynthesis, as has previously been demonstrated for periphyton communities grown on artificial, inert substrata. Thus, light/photosynthetic mediated stimulation of extracellular enzyme activities may be a common occurrence in microbial communities associated with natural decaying plant litter in wetlands and might engender diurnal patterns in other microbial decay processes (e.g., production, organic matter decomposition, and mineralization).     

Legume inoculant application methods: effects on nodulation patterns,nitrogen fixation,crop growth and yield in narrow-leaf lupin and faba bean

Aims

Plant growth forms can influence carbon cycling, particularly in carbon-rich ecosystems like northern peatlands; however, mechanistic evidence of this relationship is limited. Our aim was to determine if northern peatland plant growth forms alter belowground dissolved carbon chemistry and enhance carbon release through stimulated microbial metabolism.

Methods

We used replicated, peat monoliths populated exclusively by Sphagnum mosses, graminoids, or bare peat and quantified changes in belowground dissolved organic carbon chemistry, microbial metabolism, as well as respired CO₂.

Results

The graminoid growth form was significantly distinct in belowground dissolved organic carbon chemistry with carbon compound lability 20 % and 11 % greater than bare peat and Sphagnum moss respectively. The labile dissolved organic carbon stimulated the microbial community, as indicated by greater microbial metabolic activity and richness values in conjunction with 50 % higher respired CO₂ fluxes under the graminoid treatment.

Conclusions

Our results provide mechanistic evidence that peatland plant growth forms can drive carbon cycling processes by altering dissolved organic carbon chemistry to prompt cascading effects on the microbial community and carbon release — trends suggestive of microbial priming effects. Should climate change increase graminoid prevalence at the expense of Sphagnum moss northern peatland carbon store stability may be threatened by this mechanism.

     

Evaluating the use of dominant microbial consumers (testate amoebae) as indicators of blanket peatland restoration

Peatlands represent globally-important ecosystems and carbon stores. However, large areas of peatland have been drained for agriculture, or peat has been harvested for use as fuel or in horticulture. Increasingly, these landscapes are being restored through ditch blocking and rewetting primarily to improve biodiversity and promote peat accumulation. To date we have little knowledge of how these interventions influence the microbial communities in peatlands. We compared the responses of dominant microbial consumers (testate amoebae) to drainage ditch restoration relative to unblocked ditches in a UK upland blanket peatland (Migneint, North Wales). Two techniques were used for restoration: (i) dammed ditches with re-profiling; and (ii) dammed ditches with pools of open water behind each dam. Testate communities in the inter-ditch areas changed markedly over time and between treatments illustrating the potential of this group of organisms as indicators of blanket peatland restoration status. However, the responses of testate amoebae to peat rewetting associated with restoration were partially obscured by inter-annual variability in weather conditions through the course of the experiment. Although there was considerable variability in the response of testate amoebae communities to peatland drain blocking, there were clearly more pronounced changes in samples from the dammed and reprofiled treatments including an increase in diversity, and the appearance of unambiguous wet-indicator species in relatively high abundances (including Amphitrema stenostoma, Archerella flavum, Arcella discoides type, Difflugia bacillifera and Difflugia bacillarium). This reflects a shift towards overall wetter conditions across the site and the creation of new habitats. However, water-table was not a significant control on testate amoebae in this case, suggesting a poor relationship between water table and surface moisture in this sloping blanket peatland. Our findings highlight the potential of testate amoebae as bioindicators of peatland restoration success; however, there is a need for caution as mechanisms driving change in the microbial communities may be more complex than first assumed. Several factors need to be taken into account when implementing biomonitoring studies in peatlands including: (i) the natural variability of the peatland ecosystem under changing weather conditions; (ii) any disturbance connected with the restoration procedures; and (iii) the timescales over which the ecosystem responds to the management intervention. Our results also suggest an indicator species approach based on population dynamics may be more appropriate for biomonitoring peatland restoration than examining changes at the community level.     

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.     

Site fertility and soil water-table level affect fungal biomass production and community composition in boreal peatland forests

A substantial amount of below-ground carbon (C) is suggested to be associated with fungi, which may significantly affect the soil C balance in forested ecosystems. Ergosterol from in-growth mesh bags and litterbags was used to estimate fungal biomass production and community composition in drained peatland forests with differing fertility. Extramatrical mycelia (EMM) biomass production was generally higher in the nutrient-poor site, increased with deeper water table level and decreased along the length of the recovery time. EMM biomass production was of the same magnitude as in mineral-soil forests. Saprotrophic fungal biomass production was higher in the nutrient-rich site. Both ectomycorrhizal (ECM) and saprotrophic fungal community composition changed according to site fertility and water table level. ECM fungal community composition with different exploration types may explain the differences in fungal biomass production between peatland forests. Melanin-rich Hyaloscypha may indicate decreased turnover of biomass in nutrient-rich young peatland forest. Genera Lactarius and Laccaria may be important in nutrient rich and Piloderma in the nutrient-poor conditions, respectively. Furthermore, Paxillus involutus and Cortinarius sp. may be important generalists in all sites and responsible for EMM biomass production during the first summer months. Saprotrophs showed a functionally more diverse fungal community in the nutrient-rich site.     

Effect of water table drawdown on peatland nutrient dynamics: implications for climate change

It is anticipated that a lowering of the water table and reduced soil moisture levels in peatlands may increase peat decomposition rates and consequently affect nutrient availability. However, it is not clear if patterns will be consistent across different peatland types or within peatlands given the natural range of ecohydrological conditions within these systems. We examined the effect of persistent drought on peatland nutrient dynamics by quantifying the effects of an experimentally lowered water table position (drained for a 10-year period) on peat KCl-extractable total inorganic nitrogen (ext-TIN), peat KCl-extractable nitrate (ext-NO₃ ^?), and water-extractable ortho-phosphorus (ext-PO₄ ^3?) concentrations and net phosphorus (P) and nitrogen (N) mineralization and nitrification rates at natural (control) and drained microforms (hummocks, lawns) of a bog and poor fen near Québec City, Canada. Drainage (water table drawdown) decreased net nitrification rates across the landscape and increased ext-NO₃ ^? concentrations, but did not affect net N and P mineralization rates or ext-TIN and ext-PO₄ ^3? concentrations. We suggest that the thick capillary fringe at the drained peatland likely maintained sufficient moisture above the water table to limit the effects of drainage on microbial activity, and a 20 cm lowering of the water table does not appear to have been sufficient to create a clear difference in nutrient dynamics in this peatland landscape. We found some evidence of differences in nutrient concentrations with microforms, where concentrations were greater in lawn than hummock microforms at control sites indicating some translocation of nutrients. In general, the same microtopographic differences were not observed at drained sites. The general spatial patterns in nutrient concentrations did not reflect net mineralization/immobilization rates measured at our control or drained peatlands. Rather, the spatial patterns in nutrient availability may be regulated by differences in vegetation (mainly Sphagnum moss) cover between control and drained sites and possibly differences in hydrologic connection between microforms. Our results suggest that microform distribution and composition within a peatland may be important for determining how peatland nutrient dynamics will respond to water table drawdown in northern peatlands, as some evidence of microtopographic differences in nutrient dynamics was found.     

N-Acetylglucosaminidase activities in wetlands: a global survey

A fluorogenic model substrate, methylumbelliferyl-N-acetylglucosamine was used to measure N-acetylglucosaminidase activities in wetland soils. The enzyme activity exhibited substrate saturation around 200 M, and an approximate linearity for at least 90 min incubation time. The effects of temperature, pH, toluene, and -radiation on the enzyme were also determined. The method was applied to 32 different wetland samples from both freshwater wetlands and salt-water wetlands. Overall, the activities were the lowest in bogs and much higher in freshwater marshes and flooded grasslands. The variations of the activity were not explained by a single environmental variable. However, among peatland samples (e.g., bogs and fens) only, specific enzyme activity per organic matter exhibited a significant correlation with N-acetylglucosaminidase activity, suggesting the importance of the enzyme activity in organic matter dynamics in such systems.     

Temporal and stoichiometric patterns of algal stimulation of litter-associated heterotrophic microbial activity

1. Periphyton communities associated with submerged plant detritus contain interacting autotrophic and heterotrophic microbes, and are sites of extracellular enzymatic activity. The strength and nature of these interactions might be expected to change over time as microbial communities develop on plant litter. Microbial interactions and enzymatic activity can be altered by nutrient availability, suggesting that litter stoichiometry could also affect these phenomena.
2. We grew wetland plants under ambient and nutrient-enriched conditions to generate plant litter of differing nutrient content. In two experiments, we investigated: (1) the influence of algal photosynthesis on fungal and bacterial production and the activities of four extracellular enzymes throughout a 54-day period of microbial colonisation and growth; and (2) the influence of litter stoichiometry on these relationships.
3. Ambient and nutrient-enriched standing-dead plant litter was collected and then submerged in wetland pools to allow for natural microbial colonisation and growth. Litter samples were periodically retrieved and transported to the laboratory for experiments manipulating photosynthesis using the photosystem II inhibitor DCMU (which effectively prevents algal photosynthetic activity). Algal (¹⁴C-bicarbonate), bacterial (³H-leucine), and fungal (¹⁴C-acetate) production, and β-glucosidase, β-xylosidase, leucine aminopeptidase, and phosphatase activities (MUF- or AMC-labelled fluorogenic substrates) were measured under conditions of active and inhibited algal photosynthesis.
4. Photosynthesis stimulated overall fungal and bacterial production in both experiments, although the strength of stimulation varied amongst sampling dates. Phosphatase activity was stimulated by photosynthesis during the first, but not the second, experiment. No other enzymatic responses to short-term photosynthesis manipulations were observed.
5. Microbial communities on high-nutrient litter occasionally showed increased extracellular enzyme activity, fungal growth rates, and bacterial production compared to communities on non-enriched litter, but algal and fungal production were not affected. Litter stoichiometry had no effects on fungal, bacterial, or enzymatic responses to photosynthesis, but the mean enzyme vector analysis angle (a measure of P- versus N-acquiring enzyme activity) was positively correlated to litter N:P, suggesting that elevated litter N:P led to an increase in the relative activity of P-acquiring enzymes.
6. These results supported the hypothesis that algal photosynthesis strongly influences heterotrophic microbial activity on macrophyte leaf litter, especially that of fungi, throughout microbial community development. However, the strength of this photosynthetic stimulation does not generally depend on small differences in litter nutrient content.
7. Stimulation of microbial heterotrophs by algal photosynthesis could drive diurnal shifts in periphyton community and aquatic ecosystem function, as well as linking green (photoautotroph-based) and brown (detrital-based) food webs.

     

Effects of microtopography and water table on Sphagnum palustre L. in subtropical high mountains and implications for peatland restoration

Introduction. Human disturbance has recently led to increasingly serious destruction of Sphagnum L. wetlands in subtropical high mountains, resulting in an urgent need for wetland restoration.

Methods. Through a field experiment conducted in western Hubei Province, China, the effects of four different microtopographic types [concave surface, convex surface, concave and convex surface (CC surface), and flat surface] and water table depth (0 to ?30?cm) on three growth indicators (number of capitula, coverage and biomass) of Sphagnum palustre L. were examined. The objective was to identify the optimal hydrological conditions for S. palustre growth and thus facilitate its rapid recolonisation and restoration of these wetlands.

Key results. The results showed that different microtopographic conditions significantly influenced S. palustre growth. Among them, S. palustre in the CC surface showed the worst growth, while no significant differences existed among the other three microtopographic types. Additionally, as the water table increased, the growth of S. palustre increased, but long-term flooding impeded growth. The water table affected S. palustre growth via effects on its tissue water content.

Conclusions. Microtopographic reshaping was not essential for the success of S. palustre recolonisation, and microtopography that maintained the water table to within ?10?cm of the surface without flooding were best, independent of the microtopographic types. In addition, the growth patterns of S. palustre changed with changes in the environment, which may be related to its long-term adaptation to conditions of a lower water table.     

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Cotton (Gossypium hirsutum) seeds and Sorghum vulgare caryopses are able to incorporate CO₂ through a PEP-carboxylating enzyme (EC 4.1.1.38). The enzyme activity is optimal at pH 8.2 and is unaffected by ATP, GDP or acetyl CoA. The partially purified cotton enzyme is stimulated by inorganic phosphate with an apparent Km of 0.3 mM. The enzymes from both cultivars are inhibited by pyrophosphate, malate, and aspartate but not by succinate. Kinetic studies for Sorghum and cotton seed enzymes show apparent Km values for carbonate of 5 mM and 1.2 mM and for PEP of 36 M and 5 mM, respectively. The V_max values are 90 and 3.3 nmol min^-1 mg protein^-1, respectively.A two-fold increase in the enzyme activity from cotton seeds occurs after 2 h under laboratory germination conditions after which the activity drops sharply to 1/3 of the original activity after 5 h imbibition. No such change was observed in Sorghum caryopses enzyme. A correlation between PEP-carboxylase activity and seed vigor in both cultivars was demonstrated.Abbreviations GOT glutamicoxaloacetic-transaminase - MDH malic dehydrogenase-NADH₂ - RH relative humidity     

Non-methane biogenic volatile organic compound emissions from boreal peatland microcosms under warming and water table drawdown

Boreal peatlands have significant emissions of non-methane biogenic volatile organic compounds (BVOCs). Climate warming is expected to affect these ecosystems both directly, with increasing temperature, and indirectly, through water table drawdown following increased evapotranspiration. We assessed the combined effect of warming and water table drawdown on the BVOC emissions from boreal peatland microcosms. We also assessed the treatment effects on the BVOC emissions from the peat soil after the 7-week long experiment. Emissions of isoprene, monoterpenes, sesquiterpenes, other reactive VOCs and other VOCs were sampled using a conventional chamber technique, collected on adsorbent and analyzed by GC–MS. Carbon emitted as BVOCs was less than 1% of the CO₂ uptake and up to 3% of CH₄ emission. Water table drawdown surpassed the direct warming effect and significantly decreased the emissions of all BVOC groups. Only isoprene emission was significantly increased by warming, parallel to the increased leaf number of the dominant sedge Eriophorum vaginatum. BVOC emissions from peat soil were higher under the control and warming treatments than water table drawdown, suggesting an increased activity of anaerobic microbial community. Our results suggest that boreal peatlands could have concomitant negative and positive radiative forcing effects on climate warming following the effect of water table drawdown. The observed decrease in CH₄ emission causes a negative radiative forcing while the increase in CO₂ emission and decrease in reactive BVOC emissions, which could reduce the cooling effect induced by the lower formation rate of secondary organic aerosols, both contribute to increased radiative forcing.     

Dominance of iminopeptidase activity in the human oral bacteriumTreponema denticola ATCC 35405

Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase ofT. denticola in terms of its activity towardN-l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 M p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 mM dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis ofN-l-prolyl-2-naphthylamine was found to be very characteristic ofT. denticola ATCC 35405; out of 24 differentN-l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition ofT. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease.     

Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Impact of the plant community composition on labile soil organic carbon, soil microbial activity and community structure in semi-natural grassland ecosystems of different productivity

Aims

The main objective was to describe the effects of plant litter on SOC and on soil microbial activity and structure in extensively managed grasslands in Central Germany that vary in biomass production and plant community composition.

Methods

The decomposition of shoot and root litter was studied in an incubation experiment. Labile C and N were isolated by hot water extraction (C_HWE, N_HWE), while functional groups of microbes were identified by PLFA analysis and microbial activity was measured using a set of soil exo-enzymes.

Results

The plant community composition, particulary legume species affected SOC dynamics and below-ground microbial processes, especially via roots. This was reflected in about 20% lower decomposition of root litter in low productivity grassland soil. The C_HWE soil pool was found to be a key driver of the below-ground food web, controlling soil microbial processes.

Conclusions

Below-ground responses appear to be related to the presence of legume species, which affected the microbial communities, as well as the ratio between fungal and bacterial biomass and patterns of soil enzyme activity. Low productivity fungal-dominated grasslands with slow C turnover rates may play an important role in SOC accumulation. The approach used here is of particular importance, since associated biological and biochemical processes are fundamental to ecosystem functioning.     

Effects of land use on the structure and function of leaf‐litter microbial communities in boreal streams

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An aldonolactonase AltA from Penicillium oxalicum mitigates the inhibition of β-glucosidase during lignocellulose biodegradation

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, d-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than d-glucose and d-gluconate on β-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of d-glucono-1,5-lactone to d-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of β-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

     

Predator–prey mass ratio drives microbial activity under dry conditions in Sphagnum peatlands

Mid‐ to high‐latitude peatlands are a major terrestrial carbon stock but become carbon sources during droughts, which are increasingly frequent as a result of climate warming. A critical question within this context is the sensitivity to drought of peatland microbial food webs. Microbiota drive key ecological and biogeochemical processes, but their response to drought is likely to impact these processes. Peatland food webs have, however, been little studied, especially the response of microbial predators. We studied the response of microbial predators (testate amoebae, ciliates, rotifers, and nematodes) living in Sphagnum moss carpet to droughts, and their influence on lower trophic levels and on related microbial enzyme activity. We assessed the impact of reduced water availability on microbial predators in two peatlands using experimental (Linje mire, Poland) and natural (Forbonnet mire, France) water level gradients, reflecting a sudden change in moisture regime (Linje), and a typically drier environment (Forbonnet). The sensitivity of different microbial groups to drought was size dependent; large sized microbiota such as testate amoebae declined most under dry conditions (?41% in Forbonnet and ?80% in Linje). These shifts caused a decrease in the predator–prey mass ratio (PPMR). We related microbial enzymatic activity to PPMR; we found that a decrease in PPMR can have divergent effects on microbial enzymatic activity. In a community adapted to drier conditions, decreasing PPMR stimulated microbial enzyme activity, while in extreme drought experiment, it reduced microbial activity. These results suggest that microbial enzymatic activity resulting from food web structure is optimal only within a certain range of PPMR, and that different trophic mechanisms are involved in the response of peatlands to droughts. Our findings confirm the importance of large microbial consumers living at the surface of peatlands on the functioning of peatlands, and illustrate their value as early warning indicators of change.     

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