Why aquatic scientists should use sulfur stable isotope ratios (ẟ34S) more often.

Over the last few decades, measurements of light stable isotope ratios have been increasingly used to answer questions across physiology, biology, ecology, and archaeology. The vast majority analyse carbon (δ13C) and nitrogen (δ15N) stable isotopes as the 'default' isotopes, omitting sulfur (δ34S) due to time, cost, or perceived lack of benefits and instrumentation capabilities. Using just carbon and nitrogen isotopic ratios can produce results that are inconclusive, uncertain, or in the worst cases, even misleading, especially for scientists that are new to the use and interpretation of stable isotope data. Using sulfur isotope values more regularly has the potential to mitigate these issues, especially given recent advancements that have lowered measurement barriers. Here we provide a review documenting case studies with real-world data, re-analysing different biological topics (i.e. niche, physiology, diet, movement and bioarchaeology) with and without sulfur isotopes to highlight the various strengths of this stable isotope for various applications. We also include a preliminary meta-analysis of the trophic discrimination factor (TDF) for sulfur isotopes, which suggest small (mean -0.4 ± 1.7 ‰ SD) but taxa-dependent mean trophic discrimination. Each case study demonstrates how the exclusion of sulfur comes at the detriment of the results, often leading to very different outputs, or missing valuable discoveries entirely. Given that studies relying on carbon and nitrogen stable isotopes currently underpin most of our understanding of various ecological processes, this has concerning implications. Collectively, these examples strongly suggest that researchers planning to use carbon and nitrogen stable isotopes for their research should incorporate sulfur where possible, and that the new 'default' isotope systems for aquatic science should now be carbon, nitrogen, and sulfur.

Novel sulfur isotope analyses constrain sulfurized porewater fluxes as a minor component of marine dissolved organic matter.

Marine dissolved organic matter (DOM) is a major reservoir that links global carbon, nitrogen, and phosphorus. DOM is also important for marine sulfur biogeochemistry as the largest water column reservoir of organic sulfur. Dissolved organic sulfur (DOS) can originate from phytoplankton-derived biomolecules in the surface ocean or from abiotically "sulfurized" organic matter diffusing from sulfidic sediments. These sources differ in 34S/32S isotope ratios (δ34S values), with phytoplankton-produced DOS tracking marine sulfate (21‰) and sulfurized DOS mirroring sedimentary porewater sulfide (∼0 to -10‰). We measured the δ34S values of solid-phase extracted (SPE) DOM from marine water columns and porewater from sulfidic sediments. Marine DOMSPE δ34S values ranged from 14.9‰ to 19.9‰ and C:S ratios from 153 to 303, with lower δ34S values corresponding to higher C:S ratios. Marine DOMSPE samples showed consistent trends with depth: δ34S values decreased, C:S ratios increased, and δ13C values were constant. Porewater DOMSPE was 34S-depleted (∼-0.6‰) and sulfur-rich (C:S ∼37) compared with water column samples. We interpret these trends as reflecting at most 20% (and on average ∼8%) contribution of abiotic sulfurized sources to marine DOSSPE and conclude that sulfurized porewater is not a main component of oceanic DOS and DOM. We hypothesize that heterogeneity in δ34S values and C:S ratios reflects the combination of sulfurized porewater inputs and preferential microbial scavenging of sulfur relative to carbon without isotope fractionation. Our findings strengthen links between oceanic sulfur and carbon cycling, supporting a realization that organic sulfur, not just sulfate, is important to marine biogeochemistry.

Microbial succession and dynamics in meromictic Mono Lake, California.

Mono Lake is a closed-basin, hypersaline, alkaline lake located in Eastern Sierra Nevada, California, that is dominated by microbial life. This unique ecosystem offers a natural laboratory for probing microbial community responses to environmental change. In 2017, a heavy snowpack and subsequent runoff led Mono Lake to transition from annually mixed (monomictic) to indefinitely stratified (meromictic). We followed microbial succession during this limnological shift, establishing a two-year (2017-2018) water-column time series of geochemical and microbiological data. Following meromictic conditions, anoxia persisted below the chemocline and reduced compounds such as sulfide and ammonium increased in concentration from near 0 to ~400 and ~150 µM, respectively, throughout 2018. We observed significant microbial succession, with trends varying by water depth. In the epilimnion (above the chemocline), aerobic heterotrophs were displaced by phototrophic genera when a large bloom of cyanobacteria appeared in fall 2018. Bacteria in the hypolimnion (below the chemocline) had a delayed, but systematic, response reflecting colonization by sediment "seed bank" communities. Phototrophic sulfide-oxidizing bacteria appeared first in summer 2017, followed by microbes associated with anaerobic fermentation in spring 2018, and eventually sulfate-reducing taxa by fall 2018. This slow shift indicated that multi-year meromixis was required to establish a sulfate-reducing community in Mono Lake, although sulfide oxidizers thrive throughout mixing regimes. The abundant green alga Picocystis remained the dominant primary producer during the meromixis event, abundant throughout the water column including in the hypolimnion despite the absence of light and prevalence of sulfide. Our study adds to the growing literature describing microbial resistance and resilience during lake mixing events related to climatic events and environmental change.

Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry.

RATIONALE: Sulfur isotope analysis of organic sulfur-containing molecules has previously been hindered by challenging preparatory chemistry and analytical requirements for large sample sizes. The natural-abundance sulfur isotopic compositions of the sulfur-containing amino acids, cysteine and methionine, have therefore not yet been investigated despite potential utility in biomedicine, ecology, oceanography, biogeochemistry, and other fields. METHODS: Cysteine and methionine were subjected to hot acid hydrolysis followed by quantitative oxidation in performic acid to yield cysteic acid and methionine sulfone. These stable, oxidized products were then separated by reversed-phase high-performance liquid chromatography (HPLC) and verified via offline liquid chromatography/mass spectrometry (LC/MS). The sulfur isotope ratios (δ34 S values) of purified analytes were then measured via combustion elemental analyzer coupled to isotope ratio mass spectrometry (EA/IRMS). The EA was equipped with a temperature-ramped chromatographic column and programmable helium carrier flow rates. RESULTS: On-column focusing of SO2 in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity, allowing precise (0.1-0.3‰ 1 s.d.) δ34 S measurements of 1 to 10 µg sulfur. We validated that our method for purification of cysteine and methionine was negligibly fractionating using amino acid and protein standards. Proof-of-concept measurements of fish muscle tissue and bacteria demonstrated differences up to 4‰ between the δ34 S values of cysteine and methionine that can be connected to biosynthetic pathways. CONCLUSIONS: We have developed a sensitive, precise method for measuring the natural-abundance sulfur isotopic compositions of cysteine and methionine isolated from biological samples. This capability opens up diverse applications of sulfur isotopes in amino acids and proteins, from use as a tracer in organisms and the environment, to fundamental aspects of metabolism and biosynthesis.