Estimating Population Turnover Rates by Relative Quantification Methods Reveals Microbial Dynamics in Marine Sediment

Abstract

The difficulty involved in quantifying biogeochemically significant microbes in marine sediments limits our ability to assess interspecific interactions, population turnover times, and niches of uncultured taxa. We incubated surface sediments from Cape Lookout Bight, North Carolina, USA, anoxically at 21°C for 122 days. Sulfate decreased until day 68, after which methane increased, with hydrogen concentrations consistent with the predicted values of an electron donor exerting thermodynamic control. We measured turnover times using two relative quantification methods, quantitative PCR (qPCR) and the product of 16S gene read abundance and total cell abundance (FRAxC, which stands for "fraction of read abundance times cells"), to estimate the population turnover rates of uncultured clades. Most 16S rRNA reads were from deeply branching uncultured groups, and ∼98% of 16S rRNA genes did not abruptly shift in relative abundance when sulfate reduction gave way to methanogenesis. Uncultured and increased at the onset of methanogenesis with population turnover times estimated from qPCR at 9.7 ± 3.9 and 12.6 ± 4.1 days, respectively. These were consistent with FRAxC turnover times of 9.4 ± 5.8 and 9.2 ± 3.5 days, respectively. Uncultured , which are possibly fermentative syntrophs of methanogens, and uncultured Kazan-3A-21 archaea also increased at the onset of methanogenesis, with FRAxC turnover times of 14.7 ± 6.9 and 10.6 ± 3.6 days. Kazan-3A-21 may therefore either perform methanogenesis or form a fermentative syntrophy with methanogens. Three genera of sulfate-reducing bacteria,, , and, increased in the first 19 days before declining rapidly during sulfate reduction. We conclude that population turnover times on the order of days can be measured robustly in organic-rich marine sediment, and the transition from sulfate-reducing to methanogenic conditions stimulates growth only in a few clades directly involved in methanogenesis, rather than in the whole microbial community. Many microbes cannot be isolated in pure culture to determine their preferential growth conditions and predict their response to changing environmental conditions. We created a microcosm of marine sediments that allowed us to simulate a diagenetic profile using a temporal analog for depth. This allowed for the observation of the microbial community population dynamics caused by the natural shift from sulfate reduction to methanogenesis. Our research provides evidence for the population dynamics of uncultured microbes as well as the application of a novel method of turnover rate analysis for individual taxa within a mixed incubation, FRAxC, which stands for "fraction of read abundance times cells," which was verified by quantitative PCR. This allows for the calculation of population turnover times for microbes in a natural setting and the identification of uncultured clades involved in geochemical processes.

Evidence for a growth zone for deep subsurface microbial clades in near-surface anoxic sediments

“…The core for cell counts was taken May 28, 2013 (core 7) from the same site (34°44.482’N, 77°7.435’W), and was sectioned in 3 cm intervals. The top 3 cm of marine sediment from CLB (34.6205°N, 76.5500°W) were collected from 10 m water depth on October 2, 2013 and combined into a 2L Erlenmeyer flask that was incubated anoxically for 802 days, as reported previously (17), without any amendment of substrates.…”

“…Population turnover times for a family-level classification were calculated from relative 16S rRNA gene abundances and total cell counts as described previously (17), with the following relationship: where t d,i is the doubling time of the i th family clade, t is the elapsed time, FRA i,t is the fraction read abundance of the i th clade at time t , and C tot,t is the total number of cells at time t . The product of the fraction 16S rRNA gene abundance of a particular clade and the total cell abundance will be referred to as the FRAxC of a particular clade.…”

“…Two more cell count curves were used to test the sensitivity of turnover times to cell count profile variations. In one, cell counts from a 2012 WOR core were previously published (17), and resulted in the following curve fit over 0-10 cm: depth = 14.13 exp(−4×10 −10 x cells/ml), R 2 =0.93. In the second, cell counts were taken using WebPlotDigitizer from previously published estuarine sediments from Ashleworth Quay (33).…”

“…In the second, cell counts were taken using WebPlotDigitizer from previously published estuarine sediments from Ashleworth Quay (33). For the CLB incubations, SYBR Gold direct counts of paraformaldehyde-fixed cells were performed as described previously (17).…”

“…Like most marine sediments, they contain diverse PDNCs, and have a steady sediment deposition rate allowing a conversion between depth and time (16). As a second test, we measured the growth of these organisms in 2.2 year-long incubations of sediments from Cape Lookout Bight (CLB) without any nutrient additions so that time could be measured directly (17).…”