Dataset 2. Methylated osmolyte content of seaweeds and coastal plants from Port Phillip Bay Australia

RESEARCH HYPOTHESIS

Methylated osmolytes — DMSP, TMA, TMAO, and choline — are the primary methanogenic substrates in shallow coastal sediments receiving seaweed and plant biomass. We hypothesised that osmolyte concentrations would differ substantially between species and predict methane production potential, providing a simple chemical proxy for estimating methane emissions from different biomass types.

DATA COLLECTION

This dataset contains quantified concentrations of four methylated osmolytes in six species of seaweed and coastal plant biomass collected from Port Phillip Bay, Victoria, Australia. Three seaweeds — Ulva lactuca, Ecklonia radiata (kelp), and Ceramiales (red filamentous algae) — were collected on 2 October 2023 from five sites (Werribee, Altona, St Kilda, Elwood, Green Point). Three coastal plants — Avicennia marina (mangrove leaves), Amphibolis antarctica (seagrass), and Salicornia quinqueflora (saltmarsh) — were collected on 1 August 2024 from Hastings and Flinders.

Biomass samples were frozen immediately after collection, then crushed in liquid nitrogen and 20–50 mg extracted in 2:6:1 chloroform:methanol:water with internal standards (CHAPS, CAPS, PIPES, TRIS at 2 µM). Extracts were analysed in triplicate by HILIC-HRMS on a Vanquish Horizon–Q Exactive Plus Orbitrap, using a seven-point calibration curve. Limits of detection were: choline 0.08 µM, DMSP 1.51 µM, TMA 0.46 µM, TMAO 0.05 µM. Peak integration was performed in Skyline 24.1. All concentrations are normalised to biomass dry weight (nmol/mg).

FINDINGS AND INTERPRETATION

DMSP dominated osmolyte composition in seaweeds, particularly in Ulva, where concentrations were substantially higher than in any other species or osmolyte measured. Coastal plants showed a different composition profile, with choline and TMA as the dominant osmolytes and very low DMSP. When osmolyte data were related to methane production from companion microcosm experiments (Dataset 1), DMSP content correlated strongly with seaweed methane production (adj. R² = 0.81, p < 0.001), and summed methyl group content explained methane production at near 100% stoichiometric efficiency (adj. R² = 0.82, p < 0.001). For coastal plants, particularly mangroves, analysed osmolytes accounted for less than 10% of observed methane production, indicating that other substrates — likely methanol from pectin hydrolysis — are the dominant methanogenic precursors.

This dataset enables comparison of osmolyte profiles across species and can be used alongside Dataset 1 to evaluate osmolyte content as a predictor of methane production potential. It demonstrates that DMSP is a reliable proxy for seaweed methane potential, while osmolyte measurements alone are insufficient for coastal plants.

研究假设
甲基化渗透调节物（methylated osmolytes）——二甲基巯基丙酸内盐（DMSP）、三甲胺（TMA）、三甲胺N-氧化物（TMAO）与胆碱（choline）——是接收海藻及植物生物质的浅海海岸沉积物中的主要产甲烷底物。本研究假设，不同物种间的渗透调节物浓度存在显著差异，且可用于预测产甲烷潜力，从而为估算不同生物质类型的甲烷排放提供一种简便的化学替代指标。

数据采集
本数据集包含从澳大利亚维多利亚州菲利普港湾采集的6种海藻与海岸植物生物质中4种甲基化渗透调节物的定量浓度。3种海藻——石莼（Ulva lactuca）、辐射昆布（Ecklonia radiata，昆布）和丝藻目（Ceramiales，红色丝状藻类）——于2023年10月2日从5个采样点（Werribee、Altona、St Kilda、Elwood、Green Point）采集；3种海岸植物——白骨壤（Avicennia marina，红树林叶片）、南极两栖草（Amphibolis antarctica，海草）和五蕊盐角草（Salicornia quinqueflora，盐沼植物）——于2024年8月1日从Hastings与Flinders采样点采集。

生物质样品采集后立即冷冻，随后置于液氮中研磨，称取20~50 mg样品用体积比2:6:1的氯仿:甲醇:水混合液，辅以内标（2 µM的CHAPS、CAPS、PIPES与TRIS）进行萃取。萃取液采用亲水作用色谱-高分辨质谱（HILIC-HRMS）在Vanquish Horizon–Q Exactive Plus Orbitrap质谱仪上进行三次重复分析，并使用七点校准曲线进行定量。各物质的检出限如下：胆碱（choline）0.08 µM、二甲基巯基丙酸内盐（DMSP）1.51 µM、三甲胺（TMA）0.46 µM、三甲胺N-氧化物（TMAO）0.05 µM。峰积分采用Skyline 24.1软件完成，所有浓度均以生物质干重进行归一化（单位：nmol/mg）。

研究结果与阐释
二甲基巯基丙酸内盐（DMSP）是海藻中占主导的渗透调节物，尤其在石莼（Ulva）中，其浓度显著高于其他所有物种及所检测的其他渗透调节物。海岸植物的渗透调节物组成则呈现不同特征，以胆碱（choline）与三甲胺（TMA）为主要渗透调节物，二甲基巯基丙酸内盐（DMSP）含量极低。

将渗透调节物数据与配套微宇宙实验（数据集1）中的产甲烷量进行关联分析后发现，二甲基巯基丙酸内盐（DMSP）含量与海藻产甲烷量呈强相关性（校正决定系数adj. R²=0.81，p<0.001）；总甲基团含量则以近100%的化学计量效率解释了产甲烷量（校正决定系数adj. R²=0.82，p<0.001）。对于海岸植物，尤其是红树林，所检测的渗透调节物仅占观测产甲烷量的10%以下，表明其他底物——大概率来自果胶水解的甲醇——是主要的产甲烷前体物。

本数据集可用于跨物种渗透调节物组成的比较分析，并可与数据集1结合，用于评估渗透调节物含量作为产甲烷潜力预测指标的有效性。研究结果表明，二甲基巯基丙酸内盐（DMSP）可作为海藻产甲烷潜力的可靠替代指标，但仅依靠渗透调节物检测无法充分预测海岸植物的产甲烷潜力。