Differential incorporation of one-carbon substrates among microbial populations identified by stable isotope probing from the estuary to South China Sea

Abstract

Methanol (MOH) and monomethylamine (MMA) are two typical one-carbon (C₁) compounds found in natural environments. They play an important role in marine and atmospheric chemistry, cloud formation, and global climate. The main biological sink of MOH and MMA is rapid consumption by marine microbes. Here, field-based time-series incubations with supplemental ¹³C-labelled MOH and MMA and isotope ratio analyses were performed. A substantial difference in the MOH and MMA incorporation rates and bacterial taxa were observed between the South China Sea (SCS) and the Pearl River estuary. C₁ substrates were assimilated more quickly in the estuary than the SCS shelf where MOH and MMA had similar bio-availability. However, microbial responses to MMA may be faster than to MOH in the coastal and basin surface water of the SCS despite similar active bacterial populations. Three ecological types of bacteria, in terms of response to supplemented MOH and MMA, were identified: rapid incorporation (I, dominant C₁-incorporating group), slow incorporation (II, minor C₁-incorporating group), and no incorporation (III, C₁-non-incorporating group). Members of the families Methylophilaceae (β-Proteobacteria) and Piscirickettsiaceae (γ-Proteobacteria) belonged to type I and actively incorporated substrates in the estuary and SCS, respectively. Diverse MOH and MMA-incorporating type II bacteria were identified by stable isotope probing in the SCS, and could play a more important role in the transformation of C₁ compounds in marine environments than hitherto assumed.

Figure 1

Map of the South China Sea and sampling stations. Isobaths are used as the background and the colour bar indicates water depth. Figure 1 was produced using Ocean Data View v. 4.7.10 (Schlitzer, R., Ocean Data View, odv.awi.de, 2017).

Figure 2

Bacterial community composition at 5 m water depth at site C3 based on T-RFLP analyses in density gradient fractions produced from ultracentrifugation of DNA after four days of incubation with supplemental ¹³C-labelled MOH. (a) Relative abundance of DNA in CsCl density gradients; (b) Relative abundance of T-RFLP peaks (colour bars) in each DNA fraction; (c) Nonmetric multidimensional scaling (NMDS) ordination based on the Bray-Curtis similarities between T-RFLP fingerprinting of bacterial communities in density gradient fractions. F = T-RFLP experiment failed.

Figure 3

Nonmetric multidimensional scaling (NMDS) ordination based on Bray-Curtis dissimilarities between bacterial communities in the representative heavy (H) and light (L) fractions from all samples. Each circle represents an individual community in the NMDS charts. The number in the circle represents incubation time. The dashed line indicates the Bray-Curtis dissimilarity between two communities. The ellipse indicates a cluster. There was a statistically significant difference (ANOSIM test, P value  < 0.05) between two clusters. Incubation experiment of sample from (a) site P1 (5 m water depth); (b) site S2 (5 m water depth); (c) site S2 (69 m water depth); (d) site C3 (5 m water depth); (e) site B4 (5 m water depth); (f) site B4 (200 m water depth).

Figure 4

Dendrogram of the group average model based on Bray-Curtis similarities between the representative ¹³C-DNA communities. Colour bars indicate relative abundances of phylogenetic taxa in each library. MOH = methanol; MMA = monomethylamine; d = day.

Figure 5

Phylo·genetic tree of OTU sequences with relative abundance>1% of total sequences in either the representative heavy (H) or light (L) fractions, or in-situ samples. Relative abundances of OTUs are shown as heat maps to the right of the phylogenetic tree. Figure was produced from the Interactive Tree Of Life (iTOL, http://itol.embl.de/). E, estuary; SCS, South China Sea.

Figure 6

Models of typical bacterial ecological type response to the supplemented ¹³C-labelled substrates. Sequences were divided into nine clades (see Fig. 5). Boxplot indicates the relative abundance of OTU sequences in in-situ communities (IS), light (L), and heavy (H) fractions. The top and bottom boundaries of each box indicate 75^th and 25^th quartile values, respectively, and the black lines inside each box represent the 50^th quartile (median) values. The ends of the whiskers mark the lowest and highest values, excluding outliers (black circles).