The potential nature of “dark” dissolved organic matter in the biosphere

Dark matter, also known as hidden/missing mass or nonluminous matter, is a component of the universe that spans 90 orders of magnitude in mass, ranging from ultralight bosons (often referred to as 'fuzzy dark matter' 1 , to massive primordial black holes 2 . These concepts have raised renewed interest following the detection of gravitational waves by the Laser Interferometer Gravitational-Wave Observatory (LIGO) and Virgo, which originated from the merging of black holes several tens of times more massive than the Sun [2][3][4] , which respond to gravity, and remains invisible to light 5 . Biogeochemical scientists have to tackle a similarly puzzling issue with dark dissolved organic matter (DDOM) in surface waters 6,7 . Recently, Cai et al. 6 using ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), identified 9,141 dark DOM molecules that exhibited high-molecular-weight (HMW) and greater diversity than the classical DOM subset, analyzing 38 DOM extracts covering the continuum of the Yangtze River-to-ocean, whereas undetected peaks and bacterial nodes were considered to represent DDOM 6,7 . Notably, the HMW DDOM fraction was found to increase along this river-to-ocean continuum 3 . Other studies have shown that only 8.7 and 9.6% of the 50,942 and 48,392m/z peaks of DOM measured by FT-ICR MS in, respectively, sediments and waters of worldwide rivers, could be assigned to identifiable molecular formulae 8,9 . However, these undetected peaks derived from DOM remain elusive, primarily due to lack of reference spectra available in current databases 10 .Many of the mentioned studies, however, have not taken into account the authentic sources of DOM, specifically allochthonous (terrestrial) and autochthonous (aquatic) sources, along with their optical and chemical characteristics. This oversight may lead to misconceptions regarding the authenticity of DDOM. In particular, which fractions or components of DOM should be prioritized for consideration as DDOM candidates, and what are the key fundamental questions regarding DDOM in the biosphere that remain unresolved?In general, allochthonous DOM detected in natural waters is primarily derived from soil containing decaying terrestrial plant materials 11,12 , and is then partially transported to surface waters through surface runoff and groundwater leaching [13][14][15][16] . Allochthonous DOM is predominantly composed of humic substances (HS), which include humic acids (HA), fulvic acids (FA), and protein-like substances (PLS) (Figure 1. A-C) [17][18][19][20] .In contrast, autochthonous DOM in water originates from planktonic photosynthetic organisms (e.g. phytoplankton) via photo/microbial respiration processes [21][22][23][24] , and is primarily exported as extracellular polymeric substances (EPS) (Figure 1D) [22][23][24] . The EPS subsequently release various components of DOM, including autochthonous humic-like substances (C and M types), carbohydrates, lipids, and protein-like substances, which encompass a range of amino acids, including tryptophan-like, tyrosine-like, and phenylalanine-like substances (Figure 1E-G) 22,23,25,26 . Importantly, all soil FA and PLS fractions, as well as autochthonous DOM exhibit recognized solubility across all pH conditions, and they are highly degradable and undergo modifications when passing from inland to marine waters 13,14,16,22,23,[27][28][29][30] . In contrast, HA possess a macromolecular/supramolecular structure that is chemically, and microbially recalcitrant.They exhibit multifunctional properties, including polyfunctionality, polyelectrolyticy, size polydispersity, physical heterogeneity, and structural lability 11,12,19,[31][32][33] . These characteristics are primarily responsible for the remarkable ability of HA to form organo-mineral complexes, which contribute to the stabilization of organic C [34][35][36] and serve as essential constituents in the continuous supply of nutrients for plant and microorganism growth 11,12,33,[37][38][39][40] .The solubility and/or insolubility characteristics of HA are significant features in their behavior 11,12,20 . In principle, a decrease in the solution pH enhances the intramolecular forces (IF) of HA by increasing the protonation of their functional groups. This, in turn, reduces their electron-donating capacity in aqueous solutions 20 . Specifically, as acidity increases, the net IF become predominant, leading to enhanced intramolecular interactions among various functional groups through hydrogen bonding. This interaction can render some fractions or functional groups of HA undetectable 20 . Ultimately, all functional groups associate, resulting in the precipitation of HA from the solution 20 . For instance, as pH decreases, the concentration of alkali-extracted HA (dissolved in a 0.1 M NaOH solution at pH ~13.0) gradually diminishes, with a fraction precipitating at pH 6.0, while the remaining HA fractions completely precipitate at pH 1.0. The corresponding pH-dependent changes in fluorescence (excitation-emission matrix, EEM) spectra and their peaks (C and A) are illustrated in Figure 2 20 . The alkali-extracted, complexed state dissolved organic carbon (DOCCS) is estimated to decrease by approximately 39.1-46.4% at pH 6 and by 48.1-53.8% at pH 1. This process is accompanied by a reduction in the intensity of the HA fluorescence peak C by approximately 29.7-47.0% at pH 6, with a complete disappearance at pH 1-2 (Figure 2) 20 .Similarly, the water extracted, labile state DOC (DOCLS) decreases by approximately 48.3-49.2% at pH 6, and completely disappears at pH 1-2 (Figure 2) 20 . These results suggest that as pH decreases, the TOC analyzer is unable to detect certain fractions of HA as DOC because these fractions remain in an insoluble state. Consequently, a pH-dependent disappearance of HA from the solution occurs. This missing fraction of DOM/HA likely consists of high-molecularweight (HMW) DOM, where intramolecular interactions among functional groups impede their identification 20 . In essence, the pH-dependent behavior of soil HA involves aggregation and precipitation processes that are also influenced by the pH and salinity of seawater. The supersaturation of coastal seawater composition can lead to the settling and storage of HA fractions in the form of organo-mineral complexes, e.g., Fe-(oxy)hydroxide minerals, at coastal seawater sites [34][35][36] . Therefore, the pH-dependent soil HA fractions could be recognized as DDOM.In essence, the solid-phase extraction (SPE) method used by Cai et al. 6 is unable to recover a significant portion of the hydrophilic DOC fraction, achieving only 3-28% recovery 41 .Consequently, major DOM fractions, such as pH-dependent soil HA, are selectively excluded from the subsequent mass spectrometry analysis following SPE 41 . In particular, the SPE-based DOM extracts collected along the river-to-ocean continuum exhibit an increasing abundance of HMW components 6 , which may be associated with the rise of autochthonous protein-like and carbohydrate matter, rather than the decline of terrestrial pH-dependent soil HA fractions throughout the river-to-ocean continuum (Figure 3) 13,16,42 . Notably, HA release sequentially degraded organic molecules that were originally bound to HA through photochemical and microbial processes [42][43] . It is highly likely that these SPEbased DOM components are optically active 16,28,41,42 . Differently, the pH-dependent soil HA are optically inactive in terms of fluorescence intensity, which diminishes with decreasing pH and are not detectable through TOC analysis 20 . The discussion above suggests that the currently proposed DDOM is not substantiated by the considerations of DOM sources.Finally, the pH-dependent soil/terrestrial HA, tentatively classified as a DDOM fraction, are prevalent in important soil and sediment environments due to their long-term C stabilization and accumulation through organo-mineral complexes [34][35] . This process, in turn, contributes to soil stability and health, promoting sustainable agricultural productivity, as well as providing living habitats for various organisms. Furthermore, a portion of pH-dependent terrestrial HA might be one of the key DOM contributors to the long-term C stability in oceanic environments 13 . Additionally, a fraction of pH-dependent soil HA could be recognized as DDOM and would also be optically inactive. Undoubtedly, pH-dependent soil HA cannot be extracted by SPE-based methanol solvents due to their insoluble macromolecular and supramolecular nature 11,12,20 , leaving them uncharacterized at the molecular level. Therefore, a substantial fraction of pH-dependent soil HA, continuously produced from photosynthetically active terrestrial plants, might be classified as DDOM, i.e., dark matter in the biosphere. This soil HA fraction fundamentally plays a crucial role in soil's structural framework, serving as a primary building block of the soil matrix across the Earth's crust and facilitating C stabilization by forming organo-mineral complexes. Lastly, the connections between DDOM and the significance of sensitivity analysis in the monitoring and management of natural water resources [45][46][47] should be the focus of further studies.