Bente Aagaard Lomstein

The turnover rate of microbial cells drops steeply by increasing depth in the seabed, in accordance with the decreasing rate of organic matter mineralization. Bacterial endospore formation and germination may take place concurrently, yet their coupling to the mineralization rates during burial is poorly understood. We used high‐resolution ¹⁴ C‐dating of coring sites on the Peru Margin to show that the upper 8.5 m of sediment had been lost due to landslides at one site, thereby exposing 13,500‐yr old sediment. Dynamic transport‐reaction modeling of sulfate and methane showed that these landslides took place several thousand years ago. We discovered that microbiological and biogeochemical data from the two sites could be combined to deconvolute the effect of sediment age on microbial turnover and organic matter mineralization. A D : L amino acid racemization model showed that the mean turnover time of both microbial biomass and bacterial endospores was a few hundred years. Detailed chrono‐stratigraphy combined with microbiological and geochemical data revealed a 10,000‐fold span of anaerobic mineralization rates across 25,000 yr of sediment age. The mean power consumption per cell of the subsurface microbial community could theoretically enable the calculated production by Firmicutes of one endospore per cell per 140–1400 years, thereby maintaining a constant stock of 10 ⁶ –10 ⁷ endospores cm ⁻³ over thousands of years. These results suggest that bacterial endospores continue to be produced and to germinate deep down in the subseafloor.

The discovery of active microbial life deeply buried beneath the seafloor has opened important questions: how do microorganisms cope with extreme energy limitation, what is their metabolic activity, and how do they repair damages to essential biomolecules? We used a D:L-amino acid model to calculate microbial biomass turnover times. We used a metagenome and metatranscriptome analysis to investigate the distribution of the gene that encodes Protein-L-iso aspartate(D-aspartate) O-methyltransferase (PCMT), an enzyme which recognizes damaged L-isoapartyl and D-aspartyl residues in proteins and catalyzes their repair. Sediment was retrieved during the Integrated Ocean Drilling Program (IODP) Expedition 347 from Landsort Deep and the Little Belt in the Baltic Sea. The study covers the period from the Baltic Ice Lake ca. 13 000 years ago to the present. Our results provide new knowledge on microbial biomass turnover times and protein repair in relation to different regimes of organic matter input. For the first time, we show that the PCMT gene was widely distributed and expressed among phylogenetically diverse groups of microorganisms. Our findings suggest that microbial communities are capable of repairing D-amino acids within proteins using energy obtained from the degradation of a mixture of labile compounds in microbial necromass and more recalcitrant organic matter.

Summary: Bacterial endospores are highly abundant in marine sediments, but their taxonomic identity and ecology is largely unknown. We selectively extracted DNA from endospores and vegetative cells and sequenced 16S rRNA genes to characterize the composition of the endospore and vegetative Firmicutes communities in the sediment and water column of Aarhus Bay (Denmark). The endospore community in the sediment was dominated by the families Bacillaceae, Lachnospiraceae, Clostridiaceae and Ruminoccocaceae . These families were also represented in the vegetative community in the sediment and the endospore community in the water column. OTUs of high relative abundance in the endospore community were also represented in the vegetative Firmicutes community. Other OTUs were exclusively found in the endospore communities. This suggests that endospores accumulate in marine sediments due to passive deposition from the water column and sporulation of vegetative cells in the sediment. Some OTUs were detected in the endospore community of the water column and the vegetative community the sediment indicating that endospores deposited from the water column may germinate upon burial/deposition in the sediment. We provide novel insight into the composition of endospore communities in marine sediments and highlight their role in microbial dispersal and as a seed bank in subsurface sediments.

We investigated the impact of temperature on the microbial turnover of organic matter (OM) in a hydrothermal vent system in Guaymas Basin, by calculating microbial bio- and necromass turnover times based on the culture-independent D:L-amino acid model. Sediments were recovered from two stations near hydrothermal mounds (8 to 10⁶ cells cm^-3 within ∼5 m of sediment depth resulting in a 100-fold lower cell number at this depth than at the cold site where numbers remained constant at 10⁸ cells cm^-3 throughout the recovered sediment. There were strong indications that the drop in cell abundance was controlled by decreasing OM quality. The quality of the sedimentary OM was determined by the diagenetic indicators %T_AAC (percentage of total organic carbon present as amino acid carbon), %T_AAN (percentage of total nitrogen present as amino acid nitrogen), aspartic acid:β-alanine ratios, and glutamic acid:γ-amino butyric acid ratios. All parameters indicated that the OM became progressively degraded with increasing sediment depth, and the OM in the hydrothermal sediment was more degraded than in the uniformly cold sediment. Nonetheless, the small community of microorganisms in the hydrothermal sediment demonstrated short turnover times. The modeled turnover times of microbial bio- and necromass in the hydrothermal sediments were notably faster (biomass: days to months; necromass: up to a few hundred years) than in the cold sediments (biomass: tens of years; necromass: thousands of years), suggesting that temperature has a significant influence on the microbial turnover rates. We suggest that short biomass turnover times are necessary for maintance of essential cell funtions and to overcome potential damage caused by the increased temperature.The reduced OM quality at the hyrothemal sites might thus only allow for a small population size of microorganisms.

The study of active microbial populations in deep, energy-limited marine sediments has extended our knowledge of the limits of life on Earth. Typically, microbial activity in the deep biosphere is calculated by transport-reaction modelling of pore water solutes or from experimental measurements involving radiotracers. Here we modelled microbial activity from the degree of D:L-aspartic acid racemization in microbial necromass (remains of dead microbial biomass) in sediments up to ten million years old. This recently developed approach (D:L-amino acid modelling) does not require incubation experiments and is highly sensitive in stable, low-activity environments. We applied for the first time newly established constraints on several important input parameters of the D:L-amino acid model, such as a higher aspartic acid racemization rate constant and a lower cell-specific carbon content of sub-seafloor microorganisms. Our model results show that the pool of necromass amino acids is turned over by microbial activity every few thousand years, while the turnover times of vegetative cells are in the order of years to decades. Notably, microbial turnover times in million-year-old sediment from the Peru Margin are up to 100-fold shorter than previous estimates, highlighting the influence of microbial activities on element cycling over geologic time scales.

Cold marine sediments harbor endospores of fermentative and sulfate-reducing, thermophilic bacteria. These dormant populations of endospores are believed to accumulate in the seabed via passive dispersal by ocean currents followed by sedimentation from the water column. However the magnitude of this process is poorly understood because the endospores present in seawater were so far not identified, and only the abundance of thermophilic sulfate-reducing endospores in the seabed has been quantified. We investigated the distribution of thermophilic fermentative endospores (TFEs) in water column and sediment of Aarhus Bay, Denmark, to test the role of suspended dispersal and determine the rate of endospore deposition and abundance in the sediment. We furthermore aimed to determine the time course of reactivation of the germinating TFEs. TFEs were induced to germinate and grow by incubating pasteurized sediment and water samples anaerobically at 50°C. We observed a sudden release of the endospore component dipicolinic acid immediately upon incubation suggesting fast endospore reactivation in response to heating. Volatile fatty acids (VFAs) and H2 began to accumulate exponentially after 3.5 h of incubation showing that reactivation was followed by a short phase of outgrowth before germinated cells began to divide. Thermophilic fermenters were mainly present in the sediment as endospores because the rate of VFA accumulation was identical in pasteurized and non-pasteurized samples. Germinating TFEs were identified taxonomically by reverse transcription, PCR amplification and sequencing of 16S rRNA. The water column and sediment shared the same phylotypes, thereby confirming the potential for seawater dispersal. The abundance of TFEs was estimated by most probable number enumeration, rates of VFA production, and released amounts of dipicolinic acid during germination. The surface sediment contained ~105-106 inducible TFEs cm-3. TFEs thus outnumber thermophilic sulfate-reducing endospores by an order of magnitude. The abundance of cultivable TFEs decreased exponentially with sediment depth with a half-life of 350 years. We estimate that 6 x109 anaerobic thermophilic endospores are deposited on the seafloor per m2 per year in Aarhus Bay, and that these thermophiles represent >10% of the total endospore community in the surface sediment.

Volatile fatty acids (VFAs) are key intermediates in the anaerobic mineralization of organic matter in marine sediments. We studied the role of VFAs in the carbon and energy turnover in the sulfate reduction zone of sediments from the sub-arctic Godthåbsfjord (SW Greenland) and the adjacent continental shelf in the NE Labrador Sea. VFA porewater concentrations were measured by a new two-dimensional ion chromatography-mass spectrometry method that enabled the direct analysis of VFAs without sample pretreatment. VFA concentrations were low and surprisingly constant (4-6 µmol L-1 for formate and acetate, and 0.5 µmol L-1 for propionate) throughout the sulfate reduction zone. Hence, VFAs are turned over while maintaining a stable concentration that is suggested to be under a strong microbial control. Estimated mean diffusion times of acetate between neighboring cells were <1 second, whereas VFA turnover times increased from several hours at the sediment surface to several years at the bottom of the sulfate reduction zone. Thus, diffusion was not limiting the VFA turnover. Despite constant VFA concentrations, the Gibbs energies (Gr) of VFA-dependent sulfate reduction decreased downcore, from -28 to -16 kJ (mol formate)-1, -68 to -31 kJ (mol acetate)-1, and -124 to -65 kJ (mol propionate)-1. Thus, Gr is

A high performance liquid chromatographic (HPLC) method for fluorescence detection of dipicolinic acid (DPA), a unique component of bacterial endospores, was developed for rapid and highly sensitive quantification of endospores. DPA was pre‐column complexed to the lanthanide metal terbium (Tb³⁺). Surplus free Tb³⁺ and the Tb³⁺‐DPA complex were separated by reverse‐phase gradient chromatography and the fluorescence emission was read at 545 nm after exitation at 270 nm. The limits of quantification (LOQ) and detection (LOD) were 0.26 and 0.08 nM, respectively. The method was used to detect DPA extracted from Bacillus subtilis endospores, DPA extracted from B. subtilis endospores added to more complex substrates such as marine sediment and canned tuna and DPA extracted from indigenous endospores in a coastal marine sediment. Endospores represented 4% of the microbial community (bacterial cells + endospores) in active surface sediment from the Aarhus Bay.

Two decades of scientific ocean drilling have demonstrated widespread microbial life in deep sub-seafloor sediment, and surprisingly high microbial-cell numbers. Despite the ubiquity of life in the deep biosphere, the large community sizes and the low energy fluxes in this vast buried ecosystem are not yet understood^1,2. It is not known whether organisms of the deep biosphere are specifically adapted to extremely low energy fluxes or whether most of the observed cells are in a dormant, spore-like state³. Here we apply a new approach—the d:l-amino-acid model—to quantify the distributions and turnover times of living microbial biomass, endospores and microbial necromass, as well as to determine their role in the sub-seafloor carbon budget. The approach combines sensitive analyses of unique bacterial markers (muramic acid and D-amino acids) and the bacterial endospore marker, dipicolinic acid, with racemization dynamics of stereo-isomeric amino acids. Endospores are as abundant as vegetative cells and microbial activity is extremely low, leading to microbial biomass turnover times of hundreds to thousands of years. We infer from model calculations that biomass production is sustained by organic carbon deposited from the surface photosynthetic world millions of years ago and that microbial necromass is recycled over timescales of hundreds of thousands of years.