Microbial Community Response to Polysaccharide Amendment in Anoxic Hydrothermal Sediments of the Guaymas Basin

Abstract

Organic-rich, hydrothermal sediments of the Guaymas Basin are inhabited by diverse microbial communities including many uncultured lineages with unknown metabolic potential. Here we investigated the short-term effect of polysaccharide amendment on a sediment microbial community to identify taxa involved in the initial stage of macromolecule degradation. We incubated anoxic sediment with cellulose, chitin, laminarin, and starch and analyzed the total and active microbial communities using bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results show a response of an initially minor but diverse population of Clostridia particularly after amendment with the lower molecular weight polymers starch and laminarin. Thus, Clostridia may readily become key contributors to the heterotrophic community in Guaymas Basin sediments when substrate availability and temperature range permit their metabolic activity and growth, which expands our appreciation of the potential diversity and niche differentiation of heterotrophs in hydrothermally influenced sediments. BONCAT-FACS, although challenging in its application to complex samples, detected metabolic responses prior to growth and thus can provide complementary insight into a microbial community’s metabolic potential and succession pattern. As a primary application of BONCAT-FACS on a diverse deep-sea sediment community, our study highlights important considerations and demonstrates inherent limitations associated with this experimental approach.

Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing

“…For GH4a-ABP-sorted samples, initial Shannon Diversity was significantly lower than the bulk or depleted samples suggesting less diversity of the probe-labeled microbes. Also seen in other systems, microbes active in the presence of complex polysaccharides (e.g., cellulose) were less diverse than total cell populationss suggesting a subset of the overall community are responsible for performing a cellulolytic phenotype [ 38 , 39 ]. The increase in diversity of enriched populations at later time points could be indicative of extremely low abundance microbial community members beginning to respond more favorably to the culture condition post-sorting and causing the evenness value to increase.…”

Natural and anthropogenic carbon input affect microbial activity in salt marsh sediment

“…Following methods used in successful BONCAT-FACS experiments by Reichart et al (2020) and Krukenberg et al (2021) , bacterial and archaeal 16S rRNA genes were amplified by 36 cycles of PCR (28 cycles for rRNA gene amplification followed by 8 cycles for barcoding) using the Earth Microbiome protocol with updated 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′) primers directly in the 96-well plates containing extracted DNA from sorted cells. Briefly, into each well containing 20 μL of cell lysate, 20 μL of Invitrogen Platinum Taq II 2X Master Mix, 1 μL of each primer (final concentration 0.2 μM each), and 5 μL of nuclease free water were added.…”

“…have been reported ( Michels et al, 2021 ). BONCAT has been an effective tool for ecophysiological studies in ocean water ( Samo et al, 2014 ; Leizeaga et al, 2017 ; Sebastián et al, 2019 ), marine sediments ( Hatzenpichler et al, 2016 ; Krukenberg et al, 2021 ), geothermal habitats ( Marlow et al, 2020 ; Reichart et al, 2020 ), soils ( Couradeau et al, 2019 ), salt marsh sediments ( Marlow et al, 2021 ), the terrestrial subsurface ( McKay et al, 2022 ; Schweitzer et al, 2022 ), waste water ( Du and Behrens, 2021 ; Madill et al, 2021 ), and host-associated microbiomes ( Riva et al, 2020 ; Valentini et al, 2020 ; Taguer et al, 2021a , b ).…”