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.

Keywords: bioorthogonal non-canonical amino acid tagging; fluorescence-activated cell sorting; heterotrophic community; metabolic activity; microbial ecology; substrate analog probing.

FIGURE 1

Alpha and beta diversity. Alpha diversity (Shannon index) decreased between fractions and timepoints (A). Principal coordinate analysis showing differences in beta diversity between samples of all fractions (B) and each fraction separately (C). Env: environmental sample (inoculum), Cel: cellulose, Chi: chitin, Lam: laminarin, Sta: starch, Non: no amendment. For each treatment filled symbols show day 2 and open symbols day 5.

FIGURE 2

Community composition on class level showing relative sequence abundance (%) for taxa occurring with >1% in at least one sample. Env: environmental sample (inoculum), Cel: cellulose, Chi: chitin, Lam: laminarin, Sta: starch, Non: no amendment.

FIGURE 3

Effect of polysaccharide amendment on abundant ASVs (i.e., occurring with >3% relative sequence abundance in at least one sample). Center log ratio (clr) transformed abundance of ASVs (A). Note that clr values indicate abundance of an ASV as relative to the mean abundance of all ASVs in a sample with positive clr values indicating abundance greater than the mean abundance. Dashed line: mean clr transformed abundance of an ASV in the environmental sample (inoculum, day 0). Log₂ fold changes >1 between no amendment and each substrate treatment for each ASV (B). Gray line represents a fold change of 0 (i.e., no difference between no amendment and substrate amendment). No data are available for Lam at day 5 as replicate C was lost and analysis relied on triplicate samples. Diamonds: DNA extract, squares: cell extract, triangles: cell sort, open symbols: day 2, filled symbols: day 5. Cel: cellulose, Chi: chitin, Lam: laminarin, Sta: starch, Non: no amendment.