Coastal hypoxia, defined as dissolved oxygen levels < 62.5 µmol kg-1, has become a worldwide problem in recent decades (Diaz and Rosenberg, 2008; Helm et al., 2011). It could affect diverse life processes from genes to ecosystems, resulting in the spatial and temporal change in marine food-web structures (Breitburg et al., 2018). Coastal deoxygenation is also tightly coupled with other global issues, such as global warming and ocean acidification (Doney et al., 2012). Formation and maintenance of eutrophication-derived hypoxia in the coastal waters should reflect the interaction between physical and biogeochemical processes (Kemp et al., 2009). Generally, seasonal hypoxia occurs in the coastal ocean when strong oxygen sinks are coupled with restricted resupply during periods of strong density stratification. Termination of the event occurs with oxygen resupply when stratification is eroded by vertical mixing (Fennel and Testa, 2019).

Bacterial respiration accounts for the largest portion of aquatic oxygen consumption and is thus pivotal for the development of hypoxia and oxygen minimum zones (Williams and del Giorgio, 2005; Diaz and Rosenberg, 2008). Generally, free-living bacteria (FLB, 0.2–0.8 µm) dominate the community respiration in many parts of the ocean (Robinson and Williams, 2005; Kirchman, 2008). Compared to the FLB, the role of particle-attached bacteria (PAB, > 0.8 µm) on community respiration is less addressed, particularly in the coastal oceans. In some coastal waters, PAB can be more important than the FLB with a higher metabolic activity that might affect carbon cycle through organic matter remineralization (Garneau et al., 2009; Lee et al., 2015). PAB was found more abundant than the FLB with a higher diversity near the mouth of the Pearl River estuary (PRE) (Li et al., 2018; Liu et al., 2020; Zhang et al., 2016). An increased contribution of PAB to respiration relative to FLB can occur during the development of coastal phytoplankton bloom (Huang et al., 2018). In the Columbia River estuary, the particle-attached bacterial activity could be 10–100-fold higher than that of its free-living counterparts, leading to its dominant role in organic detritus remineralization (Crump et al., 1998). Therefore, it is crucial to assess the respiration process associated with PAB and its controlling factors in these regions to fully understand oxygen utilization in the hypoxic area with an intense supply of particulate organic matter.

There is an increasing area of seasonal hypoxia in the nearshore bottom waters of the Pearl River estuary and the adjacent northern South China Sea (NSCS) (Yin et al., 2004; Zhang and Li, 2010; Su et al., 2017). The hypoxia is generally developed at the bottom of the salt wedge where downward mixing of oxygen is restrained due to increased stratification and where there is an accumulation of eutrophication-derived organic matter due to flow convergence driven by local hydrodynamics (Lu et al., 2018). Besides physical and biogeochemical conditions, aerobic respiration is believed to be the ultimate cause of hypoxia here (Su et al., 2017). Thus, microbial respiration had been strongly related to the consumption of bulk dissolved organic carbon in the PRE hypoxia (He et al., 2014).

Phytoplankton-derived polyunsaturated aldehydes (PUAs) are known to affect marine microorganisms over various trophic levels by acting as infochemicals and/or by chemical defenses, which strengthen their potential importance in natural environments (Ribalet et al., 2008; Ianora and Miralto, 2010; Edwards et al., 2015; Franzè et al., 2018). PUAs are produced by stressed diatoms during the oxidation of membrane polyunsaturated fatty acids (PUFAs) by lipoxygenase (Pohnert, 2000) and are released from the surface of particles to the seawater by diffusion. A perennial bloom of PUA-producing diatoms near the mouth of the PRE (Wu and Li, 2016) should support the importance of PUAs relative to other phytoplankton-derived organic compounds, such as karlotoxin by dinoflagellates, cyanotoxin by cyanobacteria, and dimethylsulfoniopropionate mainly by prymnesiophytes. Besides PUAs, there are other signaling molecules that may potentially affect bacterial activities in the low-oxygen waters, such as 2-n-pentyl-4-quinolinol (PQ) and acylated homoserine lactones (AHLs). PQ could be less important here in terms of hypoxia formation as it is generally produced as antibiosis by PAB such as Alteromonas sp. to inhibit respirations of other PAB (Long et al., 2003). AHLs could also play a less important role here since the AHL-mediated quorum sensing could be constrained by a large pH fluctuation from 7.2 to 8.8 in the bottom waters of the PRE (Decho et al., 2009).

The level of PUAs in the water column is inhomogeneous, varying from sub-nanomolar offshore to nanomolar near shore (Vidoudez et al., 2011; Wu and Li, 2016; Bartual et al., 2018) and to micromolar associated with particle hotspots (Edwards et al., 2015). The strong effect of PUAs on bacterial growth, production, and respiration has been well demonstrated in laboratory studies (Ribalet et al., 2008) and field studies (Balestra et al., 2011; Edwards et al., 2015). A nanomolar level of PUAs recently reported in the coastal waters outside the PRE was hypothesized to affect oxygen depletion by promoting microbial utilization of organic matter in the bottom waters (Wu and Li, 2016). Meanwhile, the actual role of PUAs on bacterial metabolism within the bottom hypoxia remains largely unexplored.

In this study, we investigate the particle-attached bacteria within the core of the hypoxic waters by exploring the linkage between PUAs and bacterial oxygen utilization on the suspended organic particles. There are three specific questions to address here. What are the relative roles of PAB and FLB on bacterial respiration in the hypoxic waters? What are the actual levels of PUAs in the hypoxic waters? What are the responses of PAB to PUAs in the hypoxic waters? For the first question, size-fractionated bacterial respiration rates were estimated for both FLB (0.2–0.8 µm) and PAB (> 0.8 µm) in the hypoxic waters. For the second question, the concentrations of particulate and dissolved PUAs within the hypoxic waters were measured in the field. Besides, the hotspot PUA concentration associated with the suspended particles within the hypoxic waters was directly quantified for the first time using large-volume filtration and subsequent on-site derivation and extraction. For the third question, field PUA-amended incubation experiments were conducted for PAB (> 25 µm) retrieved from the low-oxygen waters. We focused on particles of > 25 µm to explore the role of PUAs on PAB associated with sinking aggregates and large suspended particles (it may not be directly comparable to other size cutoffs in the literature). The doses of PUA treatments were selected to represent the actual levels of PUA hotspots to assess the PAB responses (including bacterial abundance, respiration, production, and community composition) to the exogenous PUAs in the hypoxic waters. By synthesizing these experimental results with the change in water-column biogeochemistry, we hope to explore the underlying mechanism for particle-adsorbed PUAs influencing community structure and metabolism of PAB in the low-oxygen waters, as well as to understand its contribution to coastal deoxygenation of the NSCS shelf sea.

Hypoxia occurs if the rate of oxygen consumption exceeds that of oxygen replenishment by diffusion, mixing, and advection (Rabouille et al., 2008). The spatial mismatch between the surface chlorophyll-a maxima and the subsurface hypoxia during our estuary-to-shelf transect should indicate that the low-oxygen feature may not be directly connected to particle export by the surface phytoplankton bloom. This outcome can be a combined result of riverine nutrient input in the surface, water-column stability driven by wind and buoyancy forcing, and flow convergence for an accumulation of organic matter in the bottom (Lu et al., 2018).

Elevated concentrations of pPUAs and dPUAs near the bottom boundary of the salt wedge should reflect a sediment source of PUAs, as the surface phytoplankton above them was very low. PUA precursors such as PUFAs could be accumulated as detritus in the surface sediment near the PRE mouth during the spring blooms (Hu et al., 2006). Strong convergence at the bottom of the salt wedge could be driven by shear vorticity and topography (Lu et al., 2018). This would allow for the resuspension of small detrital particles. Improved PUA production by oxidation of the resuspended PUFAs could occur below the salt wedge as a result of enhanced lipoxygenase activity (in the resuspended organic detritus) in response to the salinity increase by the intruded bottom seawater (Galeron et al., 2018).

Direct measurement of the adsorbed PUA concentration associated with the suspended particles of > 25 µm by the method of combined large-volume filtration and on-site derivation and extraction yields a high level of ∼ 10 µmol L-1 within the hypoxic zone. This value is comparable to those previously reported in sinking particles (> 50 µm) of the open ocean using the particle volume calculated from diatom-derived marine-snow particles (Edward et al., 2015). Note that there was also a higher level of 240 µmol L-1 found in another station outside the PRE. A micromolar level of particle-adsorbed PUAs could act as a hotspot for bacteria, likely exerting important impacts as signaling molecules on microbial utilization of particulate organic matter and subsequent oxygen consumption.

It should be mentioned that various pore sizes have been used for PAB sampling in the literature. A 0.8 µm filtration was generally accepted for separating PAB (> 0.8 µm) and FLB (0.2–0.8 µm) in the ocean (Robinson and Williams, 2005; Kirchman, 2008; Huang et al., 2018; Liu et al., 2020). Other studies defined a size of > 3 µm for PAB and 0.2–3 µm for FLB in some coastal waters (Crump et al. 1998; Garneau et al., 2009; Zhang et al., 2016). Meanwhile, there were also many studies using much larger sizes of filtration for PAB: a 5 µm filter in the German Wadden Sea (Rink et al., 2003), a 10 µm filter in the Santa Barbara Channel (DeLong et al., 1993), a 30 µm filter in the Black Sea (Fuchsman et al., 2011), and a 50 µm mesh nylon net in the North Atlantic waters (Edwards et al., 2015).

The hypoxic waters below the salt wedge have high turbidity probably due to particle resuspension. High particle concentration here may explain the previous finding of a higher abundance of PAB than FLB in the same area (e.g., Li et al., 2018; Liu et al., 2020), similar to those found in the Columbia River estuary (Crump et al., 1998). Also, anaerobic bacteria and taxa preferring low-oxygen conditions were found more enriched in the particle-attached communities than their free-living counterparts in the PRE (Zhang et al., 2016). Our field measurements suggested that bacterial respiration within the hypoxic waters was largely contributed by PAB (> 0.8 µm), with FLB (0.2–0.8 µm) playing a relatively small role. Therefore, it is crucial to address the linkage between the high-density PAB and the high level of particle-adsorbed PUAs associated with the suspended particles in the low-oxygen waters.

We choose a larger pore size of 25 µm for collecting bacteria attached to sinking aggregates and large suspended particles. Firstly, it has been suggested that the microbial respiration rate can be positively related to aggregate size (Ploug et al., 2002), and thus larger PAB likely contributes more to oxygen consumption. Secondly, larger particle size can better present the PAB taxonomy according to the previous finding of the saturation of species accumulation (for the size-fractionated bacteria) when the size is greater than 20 µm (Mestre et al., 2017). Thus, the taxonomic groups of PAB caught on particles of > 25 µm should already cover those of PAB on smaller particles of 0.8–25 µm. A similar type of filtration (30 µm) has been previously applied to study PAB in the Black Sea suboxic zones (Fuchsman et al., 2011).

Interestingly, our PUA-amended experiments for PAB (> 25 µm) retrieved from the low-oxygen waters revealed distinct responses of PAB to different doses of PUA treatments with an increase in cell growth in response to low-dose PUAs (1 µmol L-1) but an elevated cell-specific metabolic activity including bacterial respiration and production in response to high-dose PUAs (100 µmol L-1). An increase in cell density of PAB by low-dose PUAs could likely reflect the stimulating effect of PUAs on PAB growth. This finding was consistent with the previous report of a PUAs level of 0–10 µmol L-1 stimulating respiration and cell growth of PAB in sinking particles of the open ocean (Edwards et al., 2015). The negligible effect of low-dose PUAs on bacterial community structure in our experiments was also in good agreement with those found for PAB from sinking particles (Edwards et al., 2015). However, we do not see the inhibitory effect of 100 µmol L-1 PUAs on PAB respiration and production previously found in the open ocean (Edward et al., 2015). Instead, the stimulating effect for high-dose PUAs on bacterial respiration and production was even stronger with ∼ 50 % increments. The bioactivity of PUAs on bacterial strains could likely arise from their specific arrangement of two double bonds and carbonyl chain (Ribalet et al., 2008). Our findings support the important role of PUAs in enhancing bacterial oxygen utilization in low-oxygen waters.

It should be mentioned that the effect of background nanomolar PUAs on free-living bacteria remains controversial, which is not our focus in this study. Previous studies suggested that 7.5 nmol L-1 of PUAs would have a different effect on the metabolic activities of distinct bacterial groups in the NW Mediterranean Sea, although bulk bacterial abundance remained unchanged (Balestra et al., 2011). In particular, the metabolic activity of γ-Pro was least affected by nanomolar PUAs, although those of Bacteroidetes and Rhodobacteraceae were markedly depressed (Balestra et al., 2011). Meanwhile, the daily addition of 1 nmol L-1 PUAs was found to not affect the bacterial abundance and community composition during a mesocosm experiment in the Bothnian Sea (Paul et al., 2012).

It is important to verify that the PUAs are not an organic carbon source but a stimulator for PAB growth and metabolism. This was supported by the fact that the inoculated PAB could not grow in the medium with 200 µmol L-1 of PUAs although they grew pretty well in the mediums with a similar amount of ALKs or PAHs. Our results support the previous findings that the density of Alteromonas hispanica was not significantly affected by 100 µmol L-1 of PUAs in the minimal medium (without any organic carbons) during laboratory experiments (Fig. 9e), where PUAs were considered to act as cofactors for bacterial growth (Ribalet et al., 2008).

Improved cell-specific metabolism of PAB in response to high-dose PUAs was accompanied by a significant shift of bacterial community structure. The group of PAB with the greatest positive responses to exogenous PUAs was the genus Alteromonas within the γ-Pro, which is well known to have a particle-attached lifestyle with rapid growth response to organic matter (Ivars-Martinez et al., 2008). This result is contradicted by the previous finding of a reduced percentage of the γ-Pro class by high-dose PUAs in the PAB of open ocean sinking particles (Edward et al., 2015). Meanwhile, previous studies suggested that different genus groups within the γ-Pro may respond distinctly to PUAs (Ribalet et al., 2008). Our result was well consistent with the previous finding of the significant promotion effect of 13 or 106 µmol L-1 PUAs on Alteromonas hispanica from the pure culture experiment (Ribalet et al., 2008). An increase in PUAs could thus confer on some of the γ-Pro (mainly special species within the genus Alteromonas, such as A. hispanica, Fig. S2b) a competitive advantage over other bacteria, leading to their population dominance on particles in the low-oxygen waters. These results provide evidence for a previous hypothesis that PUAs could shape the bacterioplankton community composition by driving the metabolic activity of bacteria with neutral, positive, or negative responses (Balestra et al., 2011).

The taxonomic composition of PAB (> 25 µm) was substantially different from that of the bulk bacteria community in the hypoxic zone (with a large increase in γ-Pro associated with particles, Fig. S2a). This result supports the previous report of γ-Pro being the most dominant clade attached to sinking particles in the ocean (DeLong et al., 1993). A broad range of species associated with γ-Pro was known to be important for quorum-sensing processes due to their high population density (Doberva et al., 2015) associated with sinking or suspended aggregates (Krupke et al., 2016). In particular, the genera of γ-Pro such as Alteromonas and Pseudomonas are well-known quorum-sensing bacteria that can rely on diverse signaling molecules to affect particle-associated bacterial communities by coordinating gene expression within the bacterial populations (Long et al., 2003; Fletcher et al., 2007).

It has been reported that the growths of some bacterial strains of the γ-Pro such as Alteromonas spp. and Pseudomonas spp. could be stimulated and regulated by oxylipins like PUAs (Ribalet et al., 2008; Pepi et al., 2017). Oxylipins were found to promote biofilm formation of Pseudomonas spp. (Martinez et al., 2016) and could serve as signaling molecules mediating cell-to-cell communication of Pseudomonas spp. by an oxylipin-dependent quorum-sensing system (Martinez et al., 2019). As PUAs are an important group of chemical cues belonging to oxylipins (Franzè et al., 2018), it is thus reasonable to expect that PUAs may also participate as potential signaling molecules for the quorum sensing among a high-density Alteromonas or Pseudomonas. A high level of particle-adsorbed PUAs occurring on organic particles in the low-oxygen water would likely allow particle specialists such as Alteromonas to regulate bacterial community structure, which could alter species richness and diversity of PAB as well as their metabolic functions such as respiration and production when interacting with particulate organic matter in the hypoxic zone. Various bacterial assemblages may have different rates and efficiencies of particulate organic matter degradation (Ebrahimi et al., 2019). Coordination amongst these PAB could be critical in their ability to thrive on the recycling of particulate organic carbon (Krupke et al., 2016) and thus likely contribute to the acceleration of oxygen utilizations in the hypoxic zone. Nevertheless, the molecular mechanism of the potential PUA-dependent quorum sensing of PAB may be an important topic for future study.

Our findings may likely apply to other coastal systems where there are large river inputs, intense phytoplankton blooms driven by eutrophication, and strong hypoxia, such as the Chesapeake Bay, the Adriatic Sea, and the Baltic Sea. For example, the Chesapeake Bay is largely influenced by river runoff with strong eutrophication-driven hypoxia during the summer as a result of increased water stratification (Fennel and Testa, 2019) and enhanced microbial respiration fueled by organic carbons produced during spring diatom blooms (Harding et al., 2015). Similar to the PRE, there was also a high abundance of γ-Pro in the low-oxygen waters of the Chesapeake Bay associated with the respiration of resuspended organic carbon (Crump et al., 2007). Eutrophication causes intense phytoplankton blooms in the coastal ocean. Sedimentation of the phytoplankton carbons will lead to their accumulation in the surficial sediment (Cloern, 2001), including PUFA compounds derived from the lipid production. Resuspension and oxidation of these PUFA-rich organic particles during summer salt-wedge intrusion might lead to high particle-adsorbed PUAs in the water column. These PUAs could likely shift the particle-attached bacterial community to consume more oxygen when degrading particulate organic matter and thus potentially contribute to the formation of seasonal hypoxia. In this sense, the possible role of PUAs on coastal hypoxia may be a byproduct of eutrophication driven by anthropogenic nutrient loading. Further studies are required to quantify the contributions from PUA-mediated oxygen loss by aerobic respiration to total deoxygenation in the coastal ocean.

In summary, we found elevated concentrations of pPUAs and dPUAs in the hypoxic waters below the salt wedge. We also found high particle-adsorbed PUAs associated with particles of > 25 µm in the hypoxic waters based on the large-volume filtration method, which could generate a hotspot PUA concentration of > 10 µmol L-1 in the water column. In the hypoxic waters, bacterial respiration was largely controlled by PAB (> 0.8 µm), with FLB (0.2–0.8 µm) only accounting for 25 %–30 % of the total respiration. Field PUA-amended experiments were conducted for PAB associated with particles of > 25 µm retrieved from the low-oxygen waters. We found distinct responses of PAB (> 25 µm) to different doses of PUA treatments with an increase in cell growth in response to low-dose PUAs (1 µmol L-1) but an elevated cell-specific metabolic activity including bacterial respiration and production in response to high-dose PUAs (100 µmol L-1). Improved cell-specific metabolism of PAB in response to high-dose PUAs was also accompanied by a substantial shift of bacterial community structure with increased dominance of the genus Alteromonas within the γ-Pro.

Based on these observations, we hypothesize that PUAs may potentially act as signaling molecules for coordination among the high-density PAB below the salt wedge, which would likely allow bacteria such as Alteromonas to thrive in degrading particulate organic matter. Very possibly, this process by changing community compositions and metabolic rates of PAB would lead to an increase in microbial oxygen utilization that might eventually contribute to the formation of coastal hypoxia.