Development of a microscale NOx- biosensor for the study of nitrogen cycling in marine sediment

Ugo Marzocchi

Research output: Other contribution


Nitrogen is a key element for life, it is an essential component of important biomolecules (e.g. amino acids) and because of its multiple oxidative states it has been widely selected during microbial evolution as a mediator for the redox processes that allow for energy transfer and conservation. Nitrogen thus exerts an important control on biomass accumulation and on the rate of energy transfer in both artificial and natural systems. Because of their relative high solubility, the inorganic forms of nitrogen (NH4+, NO2- and NO3-) tend to accumulate in aquatic environments. Surficial marine sediments can be described as nitrogen bioreactors with several redox reactions affecting inorganic nitrogen species. Here, because of the relatively high organic matter concentration and bacterial density, nitrogen is recycled at high rates within micrometer to centimeter thick horizons. The narrow stratification of organisms and processes in surface sediments motivates scientists interested in nitrogen dynamics to use extremely small tools for detecting the spatial distribution of the nitrogen species and minimize the physical disturbance of the steep concentration gradients.The NOx- (i.e. NO3- + NO2-) microscale biosensor matches these requirements. In fact, it can be constructed with a tip diameter ranging between 25 and 100 µm. Its functioning is based on the reduction of NOx- to N2O by denitrifying bacteria and the subsequent detection of N2O by means of an amperometric microsensor. The sensitivity of the biosensor can be amplified by the electrophoretic sensitivity control system (ESC) which positively polarizes the inner side of the sensor against an external reference inserted into the analyzed medium, inducing the migration of NOx- anions into the bacterial chamber. However, nowadays the widespread application of this microscale biosensor is constrained mainly because of a short lifetime caused by the fragility of some of its components. Moreover a detailed study characterizing the ESC efficiency under different condition is still missing.The aims of this thesis are: (i) to contribute to the development of the NOx- microscale biosensor by applying a sturdier ion-permeable membrane in front of the bacterial chamber and characterizing its effects on the biosensor performance under different conditions, and (ii) to apply the sensor in an investigation of the role of nitrate in the recently described microbially-mediated long distance electric coupling between redox half reactions in marine sediment.As described in the first manuscript, we optimized the membrane permeabilization procedure in order to decrease the membrane porosity and therefore to increase its sturdiness. Successively, we assessed the effect of the sturdier membrane on the sensor sensitivity under different ESC polarizations and analyte salinities. The results of our study indicated that the membrane porosity can efficiently be reduced by diluting the LiClO4 solution routinely used during the permeabilization procedure by 30%. The application of the so obtained membrane resulted in a higher efficiency of the ESC system which in turn compensated for the initial decrease in sensitivity due by the reduced membrane permeability at salinities of 0.1 and 5 gL-1 but not at 35 gL-1. Regardless of the membrane permeability, the ESC efficiency was negatively affected by an increase in analyte salinity, as the competition between dissolved anions in carrying the charge lowered the transport of nitrate into the bacterial chamber.In the second study (Manuscript II) we incubated marine sediment under anoxic seawater and by applying microsensor techniques we detected the development of a 4-8 millimeters thick zone devoid in both nitrate and sulfides. The onset of a proton consuming (higher pH) and producing (lower pH) process at the depths of nitrate reduction and sulphides oxidation, respectively, were consistent with cathodic nitrate reduction and anodic sulfide oxidation indicating an electric coupling between the half reactions. Successively, by means of a proton-electron-nitrate mass-balance, we estimated that a consistent fraction (ranging from 10 to 78%) of the total nitrate reduction was mediated by long-distance electron transport.In conclusion, the results reported and discussed in this thesis have improved our understanding of how NOx- biosensor sensitivity is governed by the permeability of the membrane and how an applied electrical charge affects the sensitivity at various salinities. This knowledge will allow for the construction of sturdier biosensors with more reproducible characteristics to be used in environmental research. Our finding that bacteria use nitrate as final electron acceptor for driving distant oxidation of sulfide by long-distance electron transport processes may have implications for many fields of microbiology spanning from microbial ecology of sediments to corrosion research and further deep into biochemistry and molecular biology.
Original languageEnglish
TypePhD Thesis
PublisherAarhus University. Department of Bioscience
Publication statusPublished - 2013
Externally publishedYes

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