Speed bumps and barricades in the carbon cycle: substrate structural effects on carbon cycling

The observation that a fraction of organic matter produced in marine systems evades the concerted efforts of microbial communities and is buried in sediments suggests that there are ‘speed bumps’ in carbon degradation pathways that impede microbially driven remineralization processes. The initial step in degradation of macromolecules, extracellular enzymatic hydrolysis, is often stated to be ‘the’ rate-limiting step in carbon remineralization. Experimental investigations described here, however, demonstrate that at least in certain cases, microbes produce extracellular enzymes on time scales of hours to tens of hours in response to substrate addition, and hydrolysis is extremely rapid. If enzymatic hydrolysis can be rapid, what factors slow or stop organic matter degradation? A lack of the correct inducer to initiate enzyme production, and/or a lack of the correct organism to produce the required enzyme, may result in a complete lack of hydrolysis in certain environments—a barricade, rather than a speed bump. Preliminary evidence supporting this hypothesis includes a comparison of polysaccharide hydrolysis in seawater and sediments, which demonstrates that the spectrum of enzymes active in seawater and sediments are fundamentally different. Furthermore, a survey of enzyme activities in surface waters from a range of locations suggests that pelagic microbial communities also differ widely in their abilities to express specific extracellular enzymes. Trans-membrane transport through porins is yet another potential location of structure-related selectivity.Our efforts to identify speed bumps and barricades are hampered by our inability to structurally characterize in sufficient detail the macromolecular structures present in marine systems. Furthermore, assessments of organic matter ‘quality’ from a chemical perspective do not necessarily accurately reflect the availability of organic carbon to microbial communities. For these communities, in fact, ‘quality’ may be a variable, which depends on the enzymatic and uptake capabilities of community members. To begin to assess substrate structure and quality from a microbial perspective, we will have to combine specific knowledge of macromolecular structures with detailed investigations of the enzymatic and transport capabilities of heterotrophic marine microbes.     

藓羽藻(Bryopsis hypnoides)核酮糖-1，5-二磷酸羧化酶／加氧酶的分离与活性测定

采用硫酸铵分部沉淀与凝胶过滤的方法，进行藓羽藻Rubisco的分离研究。结果表明，分离的藓羽藻Rubisco经SDS-聚丙烯酰胺凝胶电泳检测呈两条清晰条带，分别为Rubisco大亚基与小亚基；与菠菜相比，藓羽藻Rubisco大亚基分子量与菠菜基本相同，而小亚基较之稍大一些。藓羽藻Rubisco活力测定结果表明，Rubisco分离过程中用硫酸铵分部沉淀后活力降低许多，分离后活力有所上升，但仍比粗提液活力弱；在Rubisco活力测定过程中，藓羽藻Rubisco的活化温度与其它物种Rubisco活化的温度不同，在低温下活化效果较好。这些结果说明Rubisco的酶活力受硫酸铵的影响而且藓羽藻Rubisco相对陆地高等植物结构不稳定。     

Ein kompetitiver Immunoassay zur Bestimmung des Herbizids Fluazifop in Trink- und Grundwasser

A Competitive Immunoassay for the Determination of the Herbicide Fluazifop in Drinking Water and Groundwater A competitive solid-phase enzyme immunoassay using rabbit polyclonal antibodies was developed for the detection of the herbicide fluazifop [(RS)-2-[4-(5-trifluoromethyl-2-pyridyloxy)phenoxy]propionic acid] in drinking water and groundwater. Present regulatory limits for drinking water in Germany were taken as the critical level. The carrier protein was bovine serum albumin; horseradish peroxidase was used as marker enzyme with 3,3′,5,5′-tetramethylbenzidine as substrate. A high concentration of high-affinity antibodies in the serum, optimization of test conditions (antibody and enzyme tracer concentration, incubation time etc.), and very low cross reactivities to substances of similar structures led to a highly sensitive and specific ELISA with a detection limit below 0.1 μg/L for fluazifop as free acid. On testing the suitability of the assay's use as a screening test with one hundred drinking-water samples, the three samples which had been spiked in the laboratory were recognized as positive with respect to their fluazifop content. Confirmation by gas chromatography-mass spectrometry showed the test results of two other samples to be false positive. False negative results did not appear. The concentration was in the detection limit region of 0.1 μg/L.     

Development of an Enzyme Immunoassay for the Analysis of the Atrazine Metabolite Hydroxyatrazine Entwicklung eines Enzymimmunoassays zur Analyse des Atrazin-Metaboliten Hydroxyatrazin

A highly sensitive and specific enzyme immunoassay (EIA) is described for the detection of the atrazine metabolite hydroxyatrazine. Polyclonal antibodies were raised in rabbits by immunization with a hapten-bovine serum albumin (BSA) conjugate containing 8 hapten residues per molecule of BSA. An EIA with a horseradish peroxidase (HRP) hapten tracer was optimized in microtitre plates. A concentration of 50% B/B₀ was found at 0.10 μg/L for hydroxyatrazine. A limit of determination for hydroxyatrazine was reached at approximately 0.01 μg/L, i.e. well below the maximum concentration permitted by the EU guidelines for drinking water and the drinking water ordinance of the FRG. The assay did not require concentration or clean-up steps for drinking water or ground water samples. Validation experiments confirmed a good accuracy and precision. Hydroxyatrazine is reported to be the main atrazine metabolite found in soil samples. As organic solvents are usually employed for soil extraction, the influence of methanol as representative organic solvent on the assay was examined. Up to a concentration of 5% (v/v) methanol, the organic solvent did not affect the assay.     

A Screening Procedure for Heavy Metals in Soil Extracts by Alcohol Dehydrogenase and Urease Inhibition Eine Screening-Methode für Schwermetalle in Extrakten aus Bodenproben,basierend auf der Inhibierung der Enzyme Alkoholdehydrogenase und Urease

A screening method for heavy metals in aqueous extracts of soil is presented which is based on inhibition of the enzymes urease and alcohol dehydrogenase. The method is suitable to detect cupric and mercury ions in concentrations below 0.01 mg/L and several other heavy metal ions in 1000 fold higher concentration. It is shown that the test may be used for screening of mercury ion concentrations exceeding 0.03 mg/L in aqueous solution when copper chelators are added to the test system. The usefulness of the presented tests to detect heavy metals eluted from soil was verified with samples from ore mining waste. The concentration of copper, lead, and zinc eluted from these samples to different amount was determined by atomic absorption spectrometry and was in good agreement with the enzyme inhibition data obtained with these samples.     

Entwicklung eines Enzymimmunoassays zum Nachweis des Herbizids Mecoprop in Trink- und Grundwasser auf der Basis von Antikörpern aus den Eiern von Legehennen

Development of an Enzyme Immunoassay for the Detection of Mecoprop in Drinking Water and Groundwater Based on Antibodies Raised in Chicken Egg Yolk Antibodies against mecoprop were isolated from egg yolk of immunized hens and were used for the development of an enzyme-linked immunosorbent assay. We tested assay parameters (pH and concentration of buffer, incubation temperature, kind of enzyme tracer) to optimize the standard curve for mecoprop. By decreasing the pH, the detection limit was reached at a concentration of 0.35 μg/L mecoprop. The concentration for 50% inhibition (50% B/B₀) was 2.8 μg/L. Dichlorprop and the methyl esters of both mecoprop and dichlorprop showed high crossreactivity (165%, 400% and 233%). Antibodies against mecoprop separated from egg yolk were compared to antibodies raised in rabbit for both sensitivity and specificity. Chicken immunoglobulins were found to be less sensitive and specific than the mammalian IgG's.     

Seasonal growth in brown trout in two New Zealand streams

Brown trout Salmo trutta Linn, were marked with individual tags and recaptured throughout a year in two small Canterbury streams. The growth recorded showed a marked seasonal variation which differed in pattern in the two streams. There was a considerable difference in the growth rate between the two populations and fish transferred from the slow‐growth stream to the faster‐growth stream showed an immediate response to the change.     

Tracing of Peroxidase Activity in Humic Lake Water

An enzyme assay was developed for studies on peroxidase activities in humic lake water. 3,4-Dimethoxybenzyl alcohol (veratryl alcohol, VeraOH) was used as tracer substrate, and peroxidase (EC 1.11.1.7) activity was measured by high-performance liquid chromatography. The chemical stability of VeraOH and its application as peroxidase substrate was tested under light and dark conditions, different hydrogen peroxide (H₂O₂) concentrations and humic matter contents. VeraOH was stable under low UV radiation at in situ conditions in lake water (<0.010...0.25 kJ m^–2 d^–1), laboratory conditions (<0.05...0.30 kJ m^–2 d^–1), and low (1...100 μM) H₂O₂ concentrations. However, peroxides oxidized VeraOH above 1...10 mM H₂O₂ concentration in sterile Millipore-Q and humic lake water. Dark incubations showed little VeraOH oxidation products. The developed peroxidase assay was tested in the growth medium of Phanerochaete chrysosporium and a bacteria isolate (P.M.D. 20.4.3.1) from mesohumic lake Pääjärvi. Peroxidase activities were also measured in natural microbial communities under standard laboratory and under in situ conditions in humic lake water. Incubation times of about 5 to 12 days were usually needed to record significant (P < 0.05) peroxidase activities, in lake waters. In situ peroxidase activities varied in pelagial surface water (0...0.5 m) on a seasonal scale between 74 nmol L^–1 h^–1 and 273 nmol L^–1 (mean: 176 nmol L^–1 h^–1) and within the water column between 110 nmol L^–1 h^–1 and 800 nmol L^–1 h^–1 (mean: 500 nmol L^–1 h^–1) in polyhumic lake Mekkojärvi.