Shifts in microbial metabolism for algal-derived organic matter utilization under dynamic oxygen conditions

1.Introduction:

As human activities intensify, declining ocean oxygen has resulted in widespread hypoxia in coastal and open-ocean regions. Hypoxia is typically defined as dissolved oxygen (DO) concentrations below 63 μM (2 mg/L)(Diaz and Rosenberg, 2008).While biodiversity impacts have been well studied, the effects on marine organic carbon (OC) cycling remain poorly understood, underscoring the need to clarify OC-hypoxia interactions (Baroni et al., 2020; Diaz and Rosenberg, 2008). Estuarine and coastal regions, particularly influenced by nutrient inputs from rivers such as the Yangtze River Estuary, are experiencing significant expansion of hypoxic zones (Breitburg et al., 2018; Zhou et al., 2020; Zhu et al., 2017). The increase in hypoxic environments significantly impacts the metabolic capabilities of microorganisms in marine ecosystems, posing a considerable challenge to ecological balance (Engel et al., 2022; Jessen et al., 2017).
In oxic environments, aerobic microbial activity plays a pivotal role in the remineralization of OC (Yamuza-Magdaleno et al., 2024). How- ever, under hypoxic conditions, the dynamics of this process undergo significant alterations. Consequently, the reduction in oxygen supply leads to shifts in microbial community composition and metabolic pathways (Bertagnolli and Stewart, 2018; Han et al., 2022). Seasonal hypoxia in coastal areas exhibits a coupled variation between apparent oxygen utilization and OC (Zhu et al., 2011). Primary production re- leases labile dissolved organic carbon (LDOC), which activates hetero- trophic bacteria, enhancing the degradation of both new LDOC and pre- existing, more refractory OC- a process termed the marine priming ef- fect (Guenet et al., 2010). The eutrophication of water column and the mineralization of large amounts of OC by heterotrophic bacteria, has led to the deoxygenation of the water column, which has become a signif- icant environmental issue (Dai et al., 2023). Generally, the reduced oxygen concentration in bottom waters leads to a greater accumulation of OC in marine sediments over a broad temporal scale (Katsev and Crowe, 2015; Keil, 1995). Following the onset of hypoxia, the microbial community shifts towards a predominance of facultative and obligate anaerobes. These microbes metabolize LDOC using alternative electron acceptors, such as nitrate or sulfate. Consequently, this shift in respira- tory pathways constitutes a redox-driven form of the marine priming effect (Ragavan and Kumar, 2021). Additionally, in the presence of nutrients like ammonia, they produce extracellular enzymes capable of decomposing OC (Guenet et al., 2010). In addition, the emergence of fermentation processes, and these microbial metabolic activities allow the marine priming effect to persist even under hypoxia conditions.
In the seasonally hypoxic waters of the Yangtze River Estuary, hyp- oxia typically occurs at depths of 30-60 m in the bottom waters. Pre- vious studies have identified surface algal blooms, primarily composed of diatoms, as the primary cause of oxygen depletion. These blooms contribute over two-thirds of the biogenic organic carbon in the estuary (Wang et al., 2017; Wang et al., 2016), leading to significant oxygen consumption in the water column, accounting for approximately 70 % of the apparent oxygen utilization (Zhou et al., 2021). Eutrophication in the estuary and hypoxic zones are also associated with high abundances of diatoms, including the representative genus Thalassiosira, which is widely distributed in global oceans and exhibits a high abundance in the Yangtze River Estuary (Li et al., 2018; Liu et al., 2024a; Malviya et al., 2016; Nelson et al., 1995). Previous studies have demonstrated that under deoxygenation conditions, the growth of Thalassiosira pseudonana may benefit from energy savings and reduced photoinhibition (Chen et al., 2021). Consequently, the accumulation of diatom-derived organic matter provides a critical oxygen-consuming pathway in seasonally hypoxic waters. Therefore, seasonal deoxygenation is closely linked to the rapid metabolic response of heterotrophic microorganisms to OC supply, generally occurring within days.
In the Yangtze River Estuary, frequent seasonal hypoxia has led to the adaptation of the local microbial community to fluctuations in redox conditions(Wang et al., 2016), making it an ideal area for studying heterotrophic bacterial populations capable of degrading diatom algal- derived dissolved organic matter (ADOM). Coastal habitats with algal- derived OC are dominated by Flavobacteria, which quickly respond to carbon inputs during remineralization (Teeling et al., 2016; West et al., 2008). The growth of these heterotrophic bacteria is intimately linked with the marine priming effect, directly influencing the mineralization of fresh OC (Williams et al., 2013). However, the impact of deoxygenation-anoxia conditions on the utilization of algal-derived OC by these heterotrophic bacteria remains unclear.
Based on prior observations of microbial resilience in seasonally hypoxic systems (Liu et al., 2024b), we hypothesize that microbial communities in the Yangtze River Estuary possess metabolic plasticity under oxygen-limited conditions. Specifically, while low DO may sup- press short-term ADOM utilization, the long-term microbial carbon processing capacity will be maintained through community-level adaptation. This study, conducted in the seasonal hypoxia zone of the Yangtze River Estuary, aims to: (1) Evaluate the microbial utilization patterns of algal-derived OC under controlled deoxygenation-anoxia conditions in an incubation experiment; (2) assess the community and metabolic changes of heterotrophic bacteria in response to algal-derived OC under deoxygenation-anoxia conditions.; and (3) investigate how deoxygenation-anoxia affects the microbial processing of algal-derived OC. This study provides new insights into clarifying the limits of OC metabolism of marine heterotrophic bacteria and the transformation of OC utilization characteristics under hypoxia

2.Methods:

2.1. Acquisition of algal-derived dissolved organic matter (ADOM) and hypoxic water samples

ADOM was obtained from cultures of Thalassiosira pseudonana (CCMA.220), sourced from the Center for Collections of Marine Algae at Xiamen University, China. This genus has been previously identified in the water column of the Yangtze River Estuary and East China Sea (Jiang et al., 2015; Liu et al., 2024a). Sterilized surface seawater, collected near Xiamen, was enriched with F/2 with silicate medium (final concentra- tion of 1 %) to cultivate T. pseudonana at 25 ◦ C. Exponential phase cells were harvested using 0.7 μm GF/F filters (47 mm diameter, Whatman, UK) and subsequently lysed using an ultrasonic cell disruptor (SM-650D, Shunmatech, China) in an ice bath. The lysate was centrifuged (8000 rpm, 5 min, 4 ◦ C) and further filtered through GF/F to obtain ADOM, which was then aliquoted and stored at − 20 ◦ C. All glassware was pre- burned at 450 ◦ C for 6 h, and other materials were soaked in 2.5 % hydrochloric acid or acid-washed to remove potential OC contamination.
Bottom water samples were collected from stations A1 and A7, located in the Yangtze River Estuary (Fig. 1). Dissolved oxygen (DO) was measured in situ by CTD (Sea-Bird SBE 9, OxygenSensor SBE 43) data (A1: 31 m, 69.1 μmol/L; A7: 17 m, 100.1 μmol/L), in August 2021. The in situ hypoxic water was retrieved using shipboard CTD and immedi- ately filtered through a 20 μm mesh and 3 μm polyester fiber membranes (47 mm diameter, Millipore, USA) to remove particulates while retain- ing microbial communities. The filtered seawater was transferred to acid-washed 20 L polyethylene buckets (PC, Nalgene, USA). For the hypoxic treatment groups, high-purity nitrogen was purged to establish low DO levels, whereas no nitrogen purging was applied to the oxygen- consuming and control groups. All seawater was then incubated in the dark at room temperature for 48 h to stabilize the in situ microbial communities.

2.2. Experimental design and sample collection

To investigate microbial utilization of ADOM under hypoxic condi- tions, we established three experimental groups for each station (A1 and A7 bottom water): (1) a hypoxic incubation group (DP), (2) an oxygen- consuming group (P), and (3) a control group (C, using bottom water from A1 only) that was maintained under in situ oxic conditions. Each group consisted of three replicates (Fig. 1). After initial settling, the filtered seawater was transferred into pre-combusted 2 L airtight glass bottles. For the DP group, dissolved oxygen was removed by sparging with high-purity nitrogen gas to establish hypoxic conditions (DO: 0-5 μmol/L), while the P and C groups were not artificially deoxygenated. After adding ADOM to the DP and P groups, the initial DOC concen- tration in the incubation bottles was approximately 1000 μM—about 8 times higher than the in situ DOC concentration. Nitrogen gas was used to maintain hypoxia in the DP group at each sampling point. DO con- centrations in the incubation system were continuously monitored using in-bottle pre-attached oxygen sensor patches and optical oxygen meters (Sensor spots: SP-PSt7-YAU, PreSens, Germany) to ensure accurate DO levels. All culture bottles were kept for 7 days in a temperature- controlled (20 ◦ C) dark incubator onboard.
Triplicate subsamples were withdrawn from each incubation bottle at both stations (A1 and A7) during the 7-day incubation. Nutrients, DOC and dissolved organic nitrogen (DON) were collected at 0, 0.5, 1, 2, 5 and 7 days; cell abundance at 0, 0.5, 1, 2, 3, 5 and 7 days, DOM optical spectra at 0, 1, 2 and 7 days; bacterial community composition at 0, 2 and 7 days; DOM molecular formulas at 0 and 7 days; and DOS con- centrations at 0, 2 and 7 days. DOS was quantified as a diagnostic in- dicator of potential sulfur incorporation into DOM under hypoxic conditions. DOC and DON samples were filtered through pre-combusted GF/F glass fiber filters and transferred into 40 mL glass bottles, acidified

to pH = 2 with H3PO4. Microbial community composition samples were collected by filtering 500 mL of culture water through 0.2 μm pore size membranes (47 mm, Millipore, USA) pre-washed with Milli-Q water. Solid-phase extraction (SPE) was performed according to a previous study (Dittmar et al., 2008), using SPE columns (500 mg, Agilent Bond Elut PPL, USA) to extract DOM components and DOS concentration from the filtrate. All samples, except for microbial community molecular membranes (stored in liquid nitrogen), were preserved at − 20 ◦ C onboard.