Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on‐line monitoring

Abstract

A new on-line method for measuring acetylene reduction is described. It consists of a gas-flow cell connected to an electronic gas-mixing system and an automatic sample loop in the gas chromatograph. Alternatively, ethylene can be determined by using laser-based trace gas detection. The laser-based trace gas detection technique achieves a detection limit that is three orders of magnitude better than gas chromatography. We have applied the on-line method to the measurement of nitrogen fixation in a culture of the heterocystous cyanobacterium Nodularia spumigena and compared it with conventional batch-type incubations. Incubation of N. spumigena in the gas-flow cell resulted in very short response times with a steady-state flux of ethylene obtained within 2 min. Nitrogenase was shown to respond immediately to changes in light and oxygen. Monitoring of nitrogenase activity could be continued for several hours without having a negative impact on nitrogen fixation rates in N. spumigena. This was not the case in batch incubations, in which changes in nitrogenase activities were recorded during incubations, probably as a result of varying oxygen concentrations. It was therefore concluded that the on-line method is superior to batch incubations when rates of nitrogenase activity are to be measured. The method is suitable for natural samples (water or sediment).

Genome-wide analysis of diel gene expression in the unicellular N2-fixing cyanobacterium Crocosphaera watsonii WH 8501

“…A second tube, connected to an Agilent 6850 gas chromatograph was inserted to the headspace for gas sampling. Apart from the online incubator, nitrogenase activity was recorded according to the acetylene reduction assay as described by Staal et al (2001). The flowthrough system was covered with dense, black plastic sheets to ensure total darkness.…”

Light dependency of nitrogen fixation in a coastal cyanobacterial mat

“…Notwithstanding the importance of N 2 fixation in microbial mats, measurements of this process have suffered from a lack of detail and resolution due to the unavailability of adequate methods. Online incubation of natural samples or cultures of diazotrophic cyanobacteria has shown its superiority compared to the traditional batch incubation in closed vials (Staal et al, 2001). The online incubation is robust and keeps the sample largely unchanged due to a constant flow of a controlled gas mixture.…”

“…We used the online method as described by Staal et al (2001). Ethylene concentration was measured using a laser-based photoacoustic (LPA) trace gas detection system (te Lintel Hekkert et al, 1998) (Sensor Sense, Nijmegen, the Netherlands; http://www.sensor-sense.nl/).…”

“…Even at the lowest nitrogenase activities the concentration of ethylene was 4100 p.p.b.v., allowing for highly accurate measurements with an error of less than 1%. The mat samples were incubated in a custommade measuring cell under a continuous flow of 2 l h À1 of a gas mixture containing 20% O 2 , 70% N 2 (each containing 0.04% CO 2 ) and 10% C 2 H 2 which was prepared using electronic mass flow controllers (Staal et al, 2001). Gas mixtures and acetylene were purchased from Hoek-Loos and Messer (both in The Netherlands), respectively.…”

Evaluación de la inoculación con microorganismos fijadores de nitrógeno asimbióticos aislados de la rizósfera de Pinus patula en Colombia

“…Esto se debe, posiblemente, a que el acetileno previene la síntesis de amonio cuando permanece por mucho tiempo en el medio a concentraciones suficientes para saturar la nitrogenasa, provocando depleción de nitrógeno en las cé-lulas. Ello induce a activación y nueva síntesis de la enzima para la fijación de nitrógeno (David y Fay 1977, Staal et al 2001, Kästner y Blöchl 2005. Con base en estos hallazgos y los encontrados en esta investigación no se hace necesario prolongar la incubación con acetileno, principalmente cuando el objetivo es determinar la presencia de la enzima nitrogenasa.…”