Derecho de Petición en Colombia como un derecho fundamental

Potential denitrification activities (Smith and Tiedje 1979; Luo et al. 1996) were determined in slurry experiments in anoxic atmosphere with 10% acetylene using 250 ml flasks sealed with butyl stoppers. Fresh sub-sampled soil (25 g) was enriched with 1.4 mM KNO3 and 10 mM glucose-C and incubated at 25°C on a rotary shaker (Swip, Edmund Bühler, Hechingen). After 15, 30, and 45 min, gas samples of 1 ml volume (B-D Plastipak, Luer Lock, BD Biosciences, Heidelberg) were taken from the headspace using gas tight syringes and were analyzed by gas chromatography (2.7.2) for N2O.

2.9.2 Potential Nitrifying Activity

Potential nitrification activities were measured after Berg and Rosswall (1985) modified by Schinner et al. (1991). For this purpose, 5 g fresh soil were enriched with 20 ml 1 mM (NH4)2SO4 and 0.1 ml 1.5 M NaClO3 and incubated at 25°C for 5 h on a horizontal shaker. Afterwards, the samples were extracted with 5 ml 2 M KCl and filtered. An aliquot of 5 ml filtrate was mixed with 3 ml 0.19 M NH4Cl buffer (pH 8.5) and 2 ml dye solution (200 ml solution contain 2 g sulfanilamide, 0.1 g naphtyl-1-diethylene-dammonium-dichloride, and 20 ml conc. H3PO4) and incubated for 15 min in darkness. The generated NO2- was measured photometrically at 520 nm via a pink diazo-complex in comparison to a standard curve prepared with sodium nitrite (2.8.4).

2.9.3 MPN of Nitrate-Reducing Bacteria

The most probable number method (MPN) was used to enumerate nitrate reducers in the environmental samples. Five g of soil or fertilizer, respectively, were dispersed in 495 ml of 0.18% sodium pyrophosphate solution and subsequently 1:10 diluted with sterile 0.9% sodium chloride solution. 3 x 9 ml of the anoxic denitrifier medium were inoculated with 1 ml per dilution step, resulting in dilution series between 10-6 _{and 10}-12 _{in three replicates. The} headspace of the test tubes were immediately flushed with N2/CO2 (80:20 [vol/vol]) using the hungate technique, sealed with butyl stoppers and incubated at 25°C. Bacterial growth was verified per eye by turbidity of the medium every week. When changes in turbidity did not occur anymore in the dilution series, medium aliquots of the last tube with growth and the first tube without growth were taken to check the nitrate concentration. NO3- was analyzed by ion chromatography (IC-System S135, conductivity detector S3111, IC Modul S4260AB, autosampler S5200, Sykam, Fürstenfeldbruck) according to the modified method from Bak et al. (1991). Signals were evaluated with the Peak Simple Chromatography Data System (Model 202, Version 2.66, SRI Instruments, California, U.S.A.). Only if growth of the nitrate reducers could be confirmed by decrease of NO3-, the samples were assessed as positive. The highest three dilutions steps exhibiting growth were selected to read the combination of numbers that then were looked up in the corrected MPN table (de Man 1983) for 3 parallels. The resulting cell counts were converted to cells per g dw soil and per g fresh fertilizer, respectively. Denitrifier medium NaCl 1.0 g l-1 MgSO4 x 7 H2O 0.486 g l-1 CaCl2 x 2 H2O 0.15 g l-1 KCl 0.5 g l-1 KH2PO4 0.2 g l-1 NH4Cl 0.25 g l-1 NaNO3 0.425 g l-1

The salts listed above were dissolved in demineralized water and autoclaved in a special flask (modified after Widdel and Bak (1992) for denitrifier). After the medium had cooled down under an N2/CO2 (80/20 [vol/vol]) atmosphere, the following components were added:

NaHCO3-buffer (1M) 30 ml l-1

Vitamin B12 (5 mg Cyanocobalamin 100 ml-1_{) 1 ml l}-1

Mix of 5 vitamins * 1 ml l-1

Riboflavin (5 mg Riboflavin 100 ml-1 _{20 mM acetic acid) 1 ml l}-1 Vitamin B1 (10 mg Thiamin-hydrochloride 100 ml-1 _{25 mM 1 ml l}-1

sodium phosphate buffer pH 3,4)

2 Material and Methods 29 * Mix of 5 vitamins: Pyridoxine-dihydrochloride 15 mg 100 ml-1 Nicotinic acid 10 mg 100 ml-1 Calcium-D(+)-Pantothenate 5 mg 100 ml-1 4-Aminobenzoic acid 4 mg 100 ml-1 D(+)-biotine 1 mg 100 ml-1

** Solution of trace elements (EDTA) (Widdel and Bak 1992): Na2-EDTA 5200 mg l-1 FeSO4 x 7 H2O 2100 mg l-1 CoCl2 x 6 H2O 190 mg l-1 ZnSO4 x 7 H2O 144 mg l-1 MnCl2 x 4 H2O 100 mg l-1 Na2MoO4 x 2 H2O 36 mg l-1 H3BO3 30 mg l-1 NiCl2 x 6 H2O 24 mg l-1 CuCl2 x 2 H2O 2 mg l-1 CuSO4 x 5 H2O 29 mg l-1

After addition of the solutions, the pH-value was measured and, if necessary, adjusted with 1 M HCl or Na2CO3 to pH 7 - 7.2.

2.9.4 Basal Respiration

Determination of soil basal respiration was done using the OxiTop® _{Control BM (WTW} Weilheim) analysis system that is based on pressure drop measurement in a closed system (Conzelmann 1996; Wagner and Fink 1996). Oxygen is consumed by respiration, and the produced CO2 is absorbed by NaOH, thus creating a negative pressure. For this purpose, 100 g fresh soil were put in a 1 l preserving jar and were adjusted to approximately 63% WHC with 5 ml pure water. After two hours of pre-incubation at room temperature, a 50 ml plastic beaker filled with 50 ml 1 M NaOH was inserted into the jar without any contact to the soil sample by placing it on the loft underneath the cover. The jar was sealed gas-tightly with the special lid adapter including the measuring head equipped with a built-in pressure sensor and infrared interface. Incubation was done at 20°C for 66 h. By pointing the respective controller OC 110 at the measuring heads, the registered 360 data points over the incubation period were transferred via infrared interface to the controller and further by cable and the communication program Achat OC to the PC for evaluation. Linear pressure decrease was converted to oxygen consumption rate using the equation:

1000 * ms * T * R ) Vs v V ( b

CRO2= n − CRO2: oxygen consumption rate [mmol O2 kg-1 dw soil h-1] b: pressure drop rate [mbar h-1_{] evaluated}

by linear regression

Vnv: netto vessel volume without soil volume [l] Vs: soil volume [l]

R = 83.14 [l mbar mol-1 _K-1_{]: gas constant} T = 293 [K]

ms: soil mass in the vessel [kg]

1000: conversion factor mol to mmol

Beside continuous pressure drop measurement an end-point quantification of CO2 produced was done by titration of a 20 ml NaOH aliquot with 1 M HCl. HCl consumption was converted to the amount of absorbed CO2 by the equation:

20 50 * ) V V (

VNaOH= aliquotNaOH− HCl VNaOH: volume of neutralized 1 M NaOH through CO2 absorption [ml]

Valiquot NaOH = 20: volume of titrated aliquot [ml] VHCl: volume of used 1 M HCl by titration [ml] 20 and 50: conversion factors from titrated NaOH

aliquot to total amount of NaOH

Since 1 ml of 1 M NaOH corresponds to 1 mmol NaOH, and 1 mmol NaOH neutralizes 0.5 mmol CO2, the amount of VNaOH was subsequently devided by 2 to obtain the amount of absorbed CO2 in mmol.

2.9.5 Substrate-Induced Respiration, SIR

Substrate-induced soil respiration was conducted as described for basal respiration (2.9.4) in principle. However, 140 mg glucose-C per 100 g fresh soil were added in 5 ml water and the incubation period was shortened to 10 h. The optimum amount of glucose was found out in a preliminary test.

2.9.6 BIOLOG Substrate Utilization Test

The carbon substrate utilization test was conducted as a rapid community-level method, whose principle was described by Garland and Mills (1991), to check differences of potential metabolic diversity between heterotrophic microbial communities in the differently fertilized soils. Microplates (Kämpfer 1988) were provided by Prof. Dr. Dr.-Ing. Peter Kämpfer, Giessen. Beside utilization of sole-carbon sources also acid production was determined via tetrazolium

2 Material and Methods 31

redox dye and production of extracellular enzymes via decomposition of chromogen substrates. Microorganisms were detached from soil particles by dispersing 10 g fresh soil in 90 ml 0.18% [wt/vol] sodium pyrophosphate in a waring blendor (Breda Scientific, Cenco, Meerbusch) at low level for 30 s. After 10 min of sedimentation, the supernatant was removed, put into an ultrasonic bath (Sonorex RK 100 H, Bandelin, Berlin) for 30 s, and was diluted 1:10 with 0.9% [wt/vol] sodium chloride. Afterwards, soil extract was mixed in a ratio of 1:2 with tetrazolium redox dye (0.3% [wt/vol] iodnitrotetrazoliumchloride, INT), and was diluted 1:2 using pure water – to obtain the same soil dilution step in all wells – for verification of extracellular enzyme production, respectively. Wells were inoculated with 50 µl of the respective soil extract and sealed with a non-toxic film. Microplates were incubated at 20°C in darkness, since chromogen substrates were sensitive to light. Tests were performed in four independent replicates per manuring treatment. After 2, 3, and 5 days, plates were evaluated visually by means of color alteration. For examination, data after two incubation days were used since afterwards alterations could hardly be observed. Data analysis (Udo Jäckel, Giessen, personal communication) was performed by transforming color alteration in 0/1 matrices (Excel, Microsoft, Unterschleißheim) of each plate. Afterwards, a “master matrix” was generated for each manuring treatment to get a survey of well positions that could be considered in the following process. Only well positions with at least three similar numbers within the four replicates of one manuring treatment were labeled for further evaluation. Cluster analyses (WinSTAT® _{for Microsoft Excel) of the labeled wells in the different manuring} treatments were performed using “Ward” as agglomeration method within the respective cropping system (without and with livestock, respectively). Additionally, all eight manuring treatments were simultaneously evaluated. However, logically, only well positions that were evaluable in all treatments and replicates compared could be included into the cluster analysis.