Elsevier

Geochimica et Cosmochimica Acta

Volume 243, 15 December 2018, Pages 116-132
Geochimica et Cosmochimica Acta

Marine ammonification and carbonic anhydrase activity induce rapid calcium carbonate precipitation

https://doi.org/10.1016/j.gca.2018.09.018Get rights and content

Abstract

During Earth’s history, precipitation of calcium carbonate by heterotrophic microbes has substantially contributed to the genesis of copious amounts of carbonate sediment and its subsequent lithification. Previous work identified the microbial sulfur and nitrogen cycle as principal pathways involved in the formation of marine calcium carbonate deposits. While substantial knowledge exists for the importance of the sulfur cycle, specifically sulfate reduction, with regard to carbonate formation, information about carbonate genesis connected to the microbial nitrogen cycle is dissatisfactory. In addition to the established pathways for carbonate mineral formation, also the potential of microbial carbonic anhydrase, a carbonate-relevant, zinc-containing enzyme, is receiving currently increased attention. However, also in this field knowledge is scarce and fragmentary. Here we demonstrate microbial carbonate precipitation as a direct result of the interplay between the microbial nitrogen cycle and a microbially produced enzyme. Using Alcanivorax borkumensis as a model organism, our experiments depict precipitation of a peloidal carbonate matrix within days to weeks, induced by simultaneous ammonification and extracellular carbonic anhydrase activity. The precipitates show similar morphology, mineralogy, δ44/40Ca, and δ88/86Sr to analogs of modern carbonate peloids. The obtained Sr/Ca partition coefficient DSr showed no clear deviation from inorganic carbonate phases, indicating that microbially mediated carbonate precipitation, indeed, follows the principles of physico-chemical precipitation. The observed relative enrichment of the precipitates in zinc might help to constrain zinc variations in natural carbonate archives. Our study demonstrates that ammonification, due to intense microbial organic matter degradation, and carbonic anhydrase may play a substantial role for calcium carbonate precipitation in paleo- and recent shallow marine environments.

Introduction

The formation and burial of carbonate-bearing rocks is by far the most important mechanism for carbon removal and storage on Earth (Sun and Turchyn, 2014). Carbonate deposits account for about one-sixth of the global sedimentary rocks (Wedepohl, 1995), representing a major fraction of the global carbon storage. A significant fraction of this carbonate is of microbial origin (Gadd, 2010). To date, a number of microbial metabolic processes, such as photosynthesis, and redox reactions using nitrogen (Castanier et al., 1999) and sulfur (Sun and Turchyn, 2014) compounds, have been identified as potentially controlling the formation of microbial carbonate minerals. Microbial sulfate reduction is suspected to be largely responsible for the formation of authigenic carbonate minerals in marine sediments (Braissant et al., 2007) and stromatolites (Visscher et al., 2000), acting as an alkalinity driver (Gallagher et al., 2012). The production of carbonate minerals due to sulfate reduction is dependent on the fate of the produced hydrogen sulfide, acting as a kinetic inhibitor for the sulfate reduction in case of excess concentrations in the vicinity of the cell (Castanier et al., 2000). Under diffusive conditions, representing the vast majority of the global marine sediments, the equilibrium pH resulting from sulfate reduction ranges between 6.5–7, rather hampering carbonate mineral precipitation (Meister, 2013). Another factor significantly impacting sulfate reduction influence on the carbonate system is the iron cycle, as hydrogen sulfide readily reacts with Fe2+ ions producing FeS2.5H++2FeOH3+HS-2Fe2++S+6H2OFe2++2HS-FeS2+H+

This reaction prevents excessive build up of hydrogen sulfide lowering the kinetic barrier for microbial sulfate reduction. However, as ferric iron (Fe3+) may be not be reduced fast enough to maintain equilibrium conditions, the availability of Fe2+ might represent the kinetic bottleneck controlling iron sulfide and carbonate formation (Coleman, 1985). Therefore, the question arises, whether contributing processes to sulfate reduction can be accounted for additional authigenic carbonate precipitation in marine sediments.

One of the most important of these processes is the microbial remineralization of organic nitrogen (ammonification) (Castanier et al., 1999). While plausible theoretical concepts (Castanier et al., 1999, Riding, 2002, Zhu and Dittrich, 2016) and numerical modeling approaches (Krumins et al., 2013) exist, to date, experimental evidence for ammonification-driven carbonate precipitation is scarce (Berner, 1968). Organic nitrogen remineralization is an essential global process, occurring in the terrestrial, freshwater and marine realms (Vitousek et al., 1997). Microbial communities in shallow marine coastal sediments play a key role in driving the oceanic nitrogen cycle (Herbert, 1999). For nitrogen remineralization amino acids undergo microbial deamination, a fundamental process termed ammonification (Sylvia et al., 2005) (Reaction 3). The metabolic product of this process is ammonia (NH3) (Sylvia et al., 2005, Gruber, 2008). When dissolved in seawater about 92% of NH3 becomes protonated forming ammonium (NH4+) and hydroxide (OH–) (Johansson and Wedborg, 1980), increasing the alkalinity of the liquid phase.Ammonification:org.NNH3+H2ONH4++OH-Alkalinity

As a consequence the carbonate system is shifted towards CO32− (Zeebe and Wolf-Gladrow, 2001), facilitating carbonate mineral precipitation when supersaturation is reached.

Current research activities indicate that, in addition to metabolic processes, also extracellular enzymes are involved in the formation of carbonate precipitates under natural conditions (Li et al., 2013, Li et al., 2014). In this context, one of the key enzymes is carbonic anhydrase (CA) (Li et al., 2010, Power et al., 2016), which catalyzes the interconversion of CO2 to HCO3 and H+ (Reaction 4). The active site of this enzyme contains a zinc ion (Maren, 1967).Carbonicanhydraseactivity:CO2+H2OCAHCO3-+H+

CAs are ubiquitously present in pro- and eukaryotic organisms, participating in numerous physiological processes (Achal and Pan, 2011). The activity of CA largely governs the concentrations of intra- or extracellular CO2 and HCO3 (Nimer et al., 1994). Although the overall dissolved inorganic carbon (DIC) concentration is not changed, unless CO2 escapes from the liquid phase, the concentration of HCO3 is directly dependent on the CA activity (Achal and Pan, 2011). The production of CO32− from HCO3, a pre-requisite for the precipitation of CaCO3, is directly dependent on pH. An increase in CO32− concentration takes place under alkaline conditions (Zeebe and Wolf-Gladrow, 2001), as provided by ammonification (Reaction 5).HCO3-+NH3CO32-+NH4+

The key objects of the present study included the evaluation of microbial ammonification as a principal driver for marine carbonate mineral precipitation in an experimental approach under laboratory conditions. In addition, the role of carbonic anhydrase within a biofilm with regard to carbonate nucleation was studied by imaging of the spatial distribution of the enzyme and conducting carbonic anhydrase inhibition experiments. In order to constrain the mode of mineral precipitation and crystal growth a combination of microscopy imaging technologies was used. Mineral properties, including carbonate phase identification, as well as element and isotope geochemistry was obtained using a combination of X-ray diffraction, ion concentration measurements by inductively coupled plasma optical emission spectrometry, and thermal ionization mass spectrometer.

We demonstrate, to our knowledge for the first time, rapid carbonate precipitation as a result of combined remineralization of organic nitrogen (ammonification) and extracellular activity of the enzyme carbonic anhydrase (CA), using the benthic marine aerobic heterotrophic γ-proteobacterium Alcanivorax borkumensis SK2 (Yakimov et al., 1988) as a model organism. The combined processes concurrently increased the ambient alkalinity, pH, and the concentration of CO3, inducing supersaturation for calcium carbonate and subsequent carbonate precipitation.

Section snippets

Bacteria culturing and media composition

For all laboratory experiments the bacteria strain Alcanivorax borkumensis SK2 was used (Yakimov et al., 1988). The strain was purchased from the DSMZ (German Collection of Microorganisms and Cell Cultures, DSMZ No. 11573, Type strain).

Natural North Sea water was used as the basis of maintenance and experimental growth media. After UV-light and filter sterilization (Whatman Polycap 75 AS-GMF/Nylon 0.2 µm) the seawater was amended with peptone (5 g l−1), yeast extract (1 g l−1), Fe(III)-citrate

Crystal mineralogy and morphology

Inoculation of liquid and agar medium with A. borkumensis resulted in mineral precipitation within three days, while sterile controls remained crystal-free. The precipitates were composed of aragonite (40–60 wt%) and high-magnesium calcite (MgCO3 concentration 25 ± 6 mol%, SD) (Fig. 1). Systematic differences in morphology and mineralogy were not observed between crystals grown on agar or in liquid medium.

During the incubation time, the nucleated crystals underwent considerable changes in

Ammonification and carbonic anhydrase

Microbial carbonate precipitation in nature occurs as a consequence of either autotrophic or heterotrophic processes (Castanier et al., 2000). Heterotrophic pathways include the dissimilatory reduction of nitrate (Tiedje, 1988), sulfate reduction (Visscher et al., 2000), anaerobic methane oxidation (Greinert et al., 2001), the degradation of urea (Ferris et al., 2003), and the ammonification of amino-acids (Castanier et al., 1999). While conclusive evidence exists for the first four processes

Conclusions

The present study extends recent theories of microbial marine carbonate production and diagenesis by showing that (I) CaCO3 matrix precipitation can be microbially mediated already close to the sediment surface; (II) extracellular enzymatic activity of microbial ammonification in combination with CA can be directly involved in peloidal carbonate formation, a substantial matrix fraction of shallow marine carbonate deposits. (III) The element and isotope uptake kinetics of the microbial carbonate

Acknowledgments

We thank A. Kolevica, R. Surberg, S. Jauch, M. Thöner, and J. Oesert for laboratory assistance. For fruitful discussions M. AlKhatib is acknowledged. L. Bryant is thanked for comments on earlier drafts. This research was carried out in the framework of the CHARON Research Group (DFG Forschergruppe 1644) funded by the German Research Foundation. Further financial support was provided by the Cluster of Excellence 80 “The Future Ocean” and the GEOMAR Helmholtz Centre for Ocean Research Kiel,

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    Present Address: University of California, 595 Charles E. Young Drive, East, Los Angeles, CA 90095-1567, USA.

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