The Role of Abundant Organic Macroaggregates in Planktonic Metabolism in a Tropical Bay

Abstract

Abundant large organic aggregates, which form mucous webs up to a few decimeters in length, have been observed in Baía de Todos os Santos (BTS), northeastern Brazil. This communication presents preliminary results from field (February 2015) and laboratory (June 2015) experiments that aimed to determine preliminary values for respiration and near-maximum photosynthesis and the impact of macroaggregates on respiration rates. The experiments included the determination of respiration in controls, with the mechanical removal and addition of macroaggregates. The field experiment during a flood tide presented the lowest respiration rate (−7.0 ± 0.7 µM L⁻¹ d⁻¹), average net primary production (8.9 ± 4.5 µM L⁻¹ d⁻¹), and gross primary production (16.0 ± 10 µM L⁻¹ d⁻¹), with a ratio of gross primary production to respiration of 2.3. The control experiments during an ebb tide showed a mean respiration rate of 8.7 ± 2.3 µM L⁻¹ d⁻¹, whereas, after macroaggregate removal, this was 9.5 ± 4.5 µM L⁻¹ d⁻¹. In the laboratory experiments, the control sample respiration rate of 18.4 ± 1.4 µM L⁻¹ d⁻¹ was slightly increased to 20.6 ± 0.1 µM L⁻¹ d⁻¹ after aggregate removal. The addition of aggregates to the control sample increased the respiration rate by approximately 3-fold, to 56.5 ± 4.8 µM L⁻¹ d⁻¹. These results indicate that macroaggregates could have an important role in pelagic and benthic respiration, as well as in the whole bay’s metabolism.

1. Introduction

Abundant organic aggregates, which form mucous webs up to a few decimeters in length, have been observed in Baía de Todos os Santos Bay (BTS), a large (1283 km²), tropical, well-mixed estuary in northeast Brazil [1]. The dimensions of these aggregates are unusual in estuarine turbulent environments, where current shear slivers larger particles [2]. Although their origin has yet to be investigated, their existence could initially be ascribed to (i) bacterial action on oil from natural seeps, former submarine exploration fields, oil installations and refinery operations, as observed by Passow et al. [3], or (ii) the accumulation of phytoplanktonic transparent exopolymers, such as those in marine snow [4] and extensive mucilage production in enclosed seas [5,6]. In the context of the algal source, increased nutrient levels also significantly enhance bacterial exopolysaccharide production, contributing to microbial aggregation in marine ecosystems. These interactions are important to the understanding of harmful algal blooms and marine mucilage events [7]. Abiotic aggregation of organic matter favored by lower salinities has also been observed, with concomitant bacterial growth in this substrate [8] and increased respiration rates [9].

The BTS water is characterized by low suspended particulate matter (SPM) concentrations (mean < 10 mg/L) and relatively high contents of particulate organic carbon (POC, mean > 35%) [2,10]. The reported mean POC content in the macroaggregates was 20% at the surface and 17% at greater depths, lower than the POC content of the SPM. By sinking at speeds of at least one order of magnitude greater than regular estuarine flocs, the macroaggregates enhance vertical sediment fluxes and sedimentation rates [2], and may therefore increase the vertical fluxes of organic carbon.

In addition to influencing vertical transport processes, these macroaggregates may also play an important role in regulating planktonic metabolism in bay waters, providing a large substrate for bacteria and phytoplankton to adhere, and similarly to what occurs with marine snow, they may boost both heterotrophic and autotrophic processes [9,11].

Aggregate-associated microbial communities demonstrate higher levels of metabolism compared to free water communities [12] and increased respiration rates [9]. By increasing organic matter deposition, they confer spatial heterogeneity to primary production and respiration and could promote the formation of anoxic microzones [13,14] that alter metabolic pathways as well as inorganic nitrogen redox processes [11,15]. The coupling of carbon and nitrogen metabolism between a cyanobacteria and heterotrophic bacteria could promote the assembling of aggregations [16].

Understanding the processes involving organic macroaggregates has biogeochemical and economical relevance and implications in the management of coastal ecosystems. Eutrophication can enhance aggregate formation through excessive phytoplankton growth, increases in dissolved organic matter, and can lead to harmful algae blooms (HABs) and hypoxia [17]. Occurrences of harmful algal blooms have already been recorded in the BTS [18], followed by dissolved oxygen reduction, hypoxia, and massive fish mortality [18,19]. The deleterious decrease in dissolved oxygen concentrations due to high respiration rates is also accompanied by a decrease in pH. Water acidification influences the carbonate system, reducing the aragonite and calcite saturation state, increasing pCO₂ and leading to fluxes of CO₂ towards the atmosphere, with deleterious effects on marine organisms and aquaculture [20,21] and the global climate [22]. Low DO concentrations and pH values may act synergistically to inflict negative effects on biota [22]. A recent marine mucilage bloom disaster apparently caused the microbiological contamination of seafood by several pathogens in the Sea of Marmara [23].

Studies concerning the role of organic aggregates in respiration in estuarine waters are still scarce in the literature and, despite their large concentrations, have not yet been undertaken in BTS. An earlier article [24] investigated the influence of a different organic matter source, a high-discharge large submarine sewage outfall, on respiration in samples collected near the BTS entrance. This communication aims to present some preliminary results from primary production and respiration experiments involving organic macroaggregates and to provide insights into the apparent role of these materials in respiration rates within the BTS.

2. Materials and Methods

The field work was executed on 25 February 2015 in a central region of the BTS (~30 m deep), where the highest concentration (up to 4 mg L⁻¹) of the largest macroaggregates were detected [2] (Supplementary Materials) (Figure 1). Surface water samples were carefully collected with an acid-washed polyethylene bucket. In the field, the water temperature, salinity, pH, and dissolved oxygen were measured with a YSI 6920 v2 probe (YSI Inc., Yellow Springs, OH, USA). The amount of colored dissolved organic matter (CDOM) was determined using a handheld Aquafluor (Turner Designs, San Jose, CA, USA).

Two sets of experiments were carried out in the field on 25 February 2015. A shorter-term experiment around the peak of insolation, between 9:00 and 14:00, collected samples near the high tide (hereinafter referred to as ‘flood’). In this experiment, no attempt was made to remove the aggregates and water was slowly siphon-drained from the bucket to flasks and vessels using a Tygon tube. DO was measured in the initial (DO_i; bucket while filling the bottles) and final time of incubation (DO_f; inside the bottles) with a Hanna HI 9143 probe. The polarographic sensor was calibrated in the field at 100% saturation (air) and the measurements were salinity-compensated. Triplicate light and dark biological oxygen demand (BOD) bottles (360 mL) were used in the approximately 5 h of incubation to evaluate in situ net primary production (NPP) and respiration (R). The light bottle samples were incubated immediately after sampling and covered with an HDPE shade cloth that reduced the solar irradiance by approximately 50%. Photosynthetically active radiation was measured using a Li-Cor Li-193 sensor (~270 μmol quanta m⁻² s⁻¹). This procedure provided a near-maximum photosynthesis rate but avoided photoinhibition.

A longer-term experiment, lasting 12 h, incubated 2 sets of triplicate dark BOD bottles with water sampled at 11:30 during the ebbing tide (hereinafter referred to as ‘ebb’). This was a first attempt to evaluate the influence of the organic aggregates on respiration. While one set of triplicates was used as a control, the other set was gravity-filtered using a 45 µm mesh as a first attempt to remove the macroaggregates. It is still possible, however, that very small fragments of weakly bound macroaggregates passed through the filter.

Another experiment aimed at assessing the influence of organic aggregates on respiration was conducted in the laboratory on 9 June 2015 (Lab). The water used in the experiments was collected at the same sampling station with a polyethylene bucket and was slowly siphon-drained to a 20 L vessel using a Tygon tube. Dissolved oxygen was measured with a Hanna HI 9143 probe (Hanna Instruments Inc., Wonsocket, RI, USA).

Water was siphon-drained to 500 mL triplicate borosilicate flasks used as controls. In addition to the control set of triplicates, we prepared two separate sets, one with the addition and the other with the removal of aggregates. Additional aggregates were previously collected on a scuba dive on 29 May 2015, using a syringe placed in 50 mL Falcon tubes. The collected aggregates were allowed to settle overnight under refrigeration, were retrieved, and were kept under refrigeration until the experiment, when a volume of 0.5 mL was added to a sample set. An attempt to remove aggregates was performed by slowly filtering the sample through a 45 µm mesh using a peristaltic pump. The water samples were incubated in the dark for 6 h.

The fluxes of dissolved oxygen due to respiration and primary production in all experiments were calculated as the difference in concentrations between the initial and final times divided by the incubation time. Dark bottles were used to determine the respiration rates (R):

$$R=-\Delta D{O}_{dark}/\Delta t$$

(1)

where ΔDO_darl is the change in dissolved oxygen concentration from the initial to final time of incubation (DO_i − DO_f) in the dark bottles (µmol L⁻¹) and Δt is the duration of incubation (h), while light bottles provided the net primary production (primary production minus respiration, NPP):

$$NPP=\Delta D{O}_{light}/\Delta t$$

(2)

where ΔDO_light is the change in dissolved oxygen concentration from the initial and final time of incubation (DO_i − DO_f) in the light bottles (µmol L⁻¹) and Δt is the duration of incubation (h).

The algebraic sum of the primary production and respiration rates (light and dark bottles) represents the gross production rate (GPP):

$$GPP=\left(\Delta D{O}_{light}-\Delta D{O}_{dark}\right)/\Delta t$$

(3)

These rates were expressed in µM L⁻¹ d⁻¹ using a daylight period of 11.4 h. The GPP/R ratio, which expresses the balance between organic matter production and consumption (autotrophy/heterotrophy), was also calculated.

The non-parametric Mann–Whitney and Brunner–Munzel tests were applied due to the small sample size, to compare the differences between the distributions. The statistical tests were performed with PAST^© software v. 4.17 (https://www.nhm.uio.no/english/research/resources/past/downloads/, accessed on 10 July 2024) [25].

3. Results

The results are shown in

Table 1. Results of variables measured in the water used in the experiments, at the initial time (i) and final (f) time, showing the respiration (R), net primary production (NPP), and gross primary production (GPP) rates.

	Laboratory			Field
Variable	Control	Added	Removed	Control Flood	Control Ebb	Ebb Removed
S	31.8	31.8	31.8	36.9	34.4	34.4
pH	7.51	7.51	7.51	8.16	7.28	7.28
CDOMi ppb	18.2	19.9	19.9	6.2	9.7	9.7
CDOMf ppb	18.4 ± 1.1	20.4 ± 0.8	18.5 ± 0.3	-	-	-
Ti °C	22.3 ± 0.8	22.3 ± 0.8	22.3 ± 0.8	28.3	27.5	27.5
Tf °C	22.5 ± 0.1	22.5 ± 0.1	22.8 ± 0.5	26.4 ± 0.3	26.8 ± 0.2	26.8 ± 0.3
DOi μM	216	216	216	196 ± 0.0	199 ± 0.5	199 ± 0.0
DOf μM	211 ± 0.4	201 ± 1.3	211 ± 0.0	193 ± 0.6	194 ± 2.5	194 ± 1.1
DOi%	97.0 ± 0.0	97.0 ± 0.0	97.0 ± 0.0	97.1 ± 0.0	97.8 ± 0.0	97.9 ± 0.2
DOf%	93.6 ± 0.2	89.0 ± 0.6	93.9 ± 0.8	93.6 ± 0.3	93.9 ± 0.5	93.7 ± 1.6
R	18.4 ± 1.4	56.5 ± 4.8	20.6 ± 0.1	7.0 ± 0.7	8.7 ± 2.3	9.5 ± 4.5
NPP	-	-	-	8.9 ± 4.5	-	-
GPP	-	-	-	16.0 ± 9.6	-	-

. The water salinity and CDOM concentrations at the beginning of the field experiments were 36.9 psu and 6.2 ppb in the flood, and 34.4 psu and 9.7 ppb in the ebbing tide, respectively. The water used in the laboratory experiments presented a lower salinity (31.8 psu) and higher CDOM concentration (18.2 ppb). The water was slightly undersaturated, with the dissolved oxygen saturation (DO%) greater than 97% at the beginning of the experiments in the laboratory.

The short-term field experiment during flood tide presented the lowest mean respiration rate among all the experiments, at 7.0 (±0.7) µM L⁻¹ d⁻¹. The mean NPP and GPP were 8.9 (±4.5) µM L⁻¹ d⁻¹ and 16.0 (±10) µM L⁻¹ d⁻¹, respectively, with a gross primary production-to-respiration ratio (GPP/R) of 2.3.

Figure 2 summarizes the results of the respiration experiments available in the BTS, as well as the results from [24], who also used DO changes on non-diluted water samples in BOD bottles incubated in the dark to measure respiration (BOD). The mean respiration rate for the flooding experiment was 7.0 (±0.7) µM L⁻¹ d⁻¹, while the mean rates for ebbing control and attempted macroaggregate-removed samples were 8.7 (±2.3) and 9.5 (±4.5) µM L⁻¹ d⁻¹, respectively.

In the laboratory, the respiration rates of the aggregate-enriched samples (56.5 ± 4.8 µM L⁻¹ d⁻¹) were approximately 3 times greater than those of the control samples (18.4 ± 1.4 µM L⁻¹ d⁻¹) and the samples that underwent aggregate removal (20.6 ± 0.1 µM L⁻¹ d⁻¹). The Mann–Whitney test results showed that there was a trend toward a difference between the treatments, but it did not reach statistical significance (p = 0.0765). Mann–Whitney’s U = 0 and Brunner–Munzel p = 0 reinforces that there was no overlap (all values of control respiration were lower).

4. Discussion

The field experiments were conducted at the end of the summer (February), when the water temperature and salinity are the highest [10,26], which is also conducive to DO concentrations below 100% saturation. Conversely, the water sample taken for the lab experiment was obtained in the climatological wettest month of the year (June), when fluvial input from peripheral catchments is highest, bringing more nutrients and dissolved organic matter (increased CDOM). This explains why the respiration rates of the control and gravity-filtered samples were higher in the laboratory despite the lower temperature. The respiration rates in the gravity-filtered samples in the field were also higher than those in the control. This can indicate the high activity of free-living bacteria in pelagic metabolism, a phenomenon also mentioned by [24] to explain the lower DO concentrations in shelf waters polluted by sewage effluent entering the bay during flood tide.

The respiration measured in the control samples collected in the flooding and ebbing tide were lower than those observed by [24] (Figure 2), which varied between 50 and 120 µM L⁻¹ d⁻¹ (see sampling sites in Figure 1). These rates were, for instance, lower than the minimum range of bacterial respiration found by Gunther et al. [27] (12 to 98 µM L⁻¹ d⁻¹) in the polluted Recife harbor and comparable to those from a eutrophicated bay in Western Australia [28] (8.81 ± 0.76 µM L⁻¹ d⁻¹). It was, however, higher than those found in unpolluted sites in Northern Australia [29] (4.85 ± 0.21 µM L⁻¹ d⁻¹) and just above the lower range (1.4 to 50 µM L⁻¹ d⁻¹) of oligotrophic regions of Southern Brazil with prevailing heterotrophy (Baía de Ilha Grande) [30]. These low respiration rates inside the BTS can explain the apparent paradox that [24] observed near the bay mouth, where lower BOD occurs at low rather than at high water. Prichett et al. [31] also observed this behavior in the lower Patuxent River estuary, an upstream member of Chesapeake Bay that receives organic-rich water from the Mid-Chesapeake during flooding. It is important to note, however, that the respiration rates in the field experiment were higher in the ebbing than flooding tide, which indicates the influence of organic matter from sources located further upstream, where the macroaggregate concentrations are higher. The rates measured in the laboratory, with a sample with lower salinity and higher CDOM, were comparable to those reported for polluted and heterotrophic locations.

On the other hand, the respiration rates from samples with aggregate addition were similar to those found by [24] for samples collected near the mouth bar exposed to the sewage outfall (Roth 1 and 2—Figure 1 and Figure 2). The vertical flux of aggregates inside the bay may thus contribute to enhancing benthic respiration rates.

The GPP/R values from our samples were high considering that dissolved oxygen subsaturation prevails in the bay most of the time [10], as was also observed in this study. The fact that NPP was measured in a surface sample in conditions near the maximum photosynthesis rate explain this result. Lower photosynthesis rates should prevail in the surface water, and higher respiration rates should exist in the bottom water [20], where the particulate organic content in the aggregates is greater [2]. The subsaturation indicates net heterotrophy, where NPP is lower than R and the rate of reaeration and vertical exchange is insufficient to compensate for the apparent oxygen consumption. Our GPP/R was near the upper range found in oligotrophic Baía de Ilha Grande [30] (1.3–2.9) and close to the mean in the strongly autotrophic coastal waters of northern Australia [29] (2.4) and greater than those found in a eutrophicated bay in Western Australia (Matilda Bay) [28] (1.9). Our results for primary production are below those listed by an estuarine–coastal inventory and are characteristic of oceanic oligotrophic waters [32].

The observed higher respiration rates in our gravity-filtered samples (ebbing tide) suggest that we failed to completely remove the aggregates. Though this explanation was not supported by microscopy or dissolved organic matter fractionation, their fragile structure apparently could not bear pressure while passing through the funnel net, which could result in leachate with more dissolved and lower size fragments. This leachate could offer a greater surface area for attaching bacteria [11]. The characterization of aggregate size distribution and this methodological limitation should be addressed in future studies by microscopy, particle size analysis, or particle counting techniques.

The higher respiration rates observed in the ebbing compared to the flood tide field experiment could be caused by the higher organic content, as expressed by the CDOM concentration. This could also reveal a somewhat refractory nature of the macroaggregates and imply that they are important for community respiration on a time scale greater than days. The high variability observed in the respiration rates of the control collected during the ebbing compared to that during flood tide in the field experiments can be explained by the size of the aggregates. The organic macroaggregates present in the sampling bucket were larger and had a darker brown coloration than those collected in the morning in the flooding tide sampling. The aggregate-enriched samples in the laboratory experiments showed the same appearance. Based on this visual inspection, we hypothesize that the difference in the size of the macroaggregates is responsible for the higher variability.

5. Conclusions

The respiration rates in the control samples in all of the experiments were lower than those observed in a previous study near the bay mouth, which is influenced by a sewage outfall. The control samples in both the flooding and ebbing tide field experiment had low respiration rates comparable to those at unpolluted sites in other regions, but in the laboratory, the water with lower salinity and higher CDOM the values were near the upper range for polluted and net heterotrophic locations. These results indicate that the rates of respiration reach higher values in response to freshwater inflow. The results of the laboratory experiments in which aggregates were added showed similar respiration rates to those in polluted shelf waters entering the bay, indicating that the abundance of macroaggregates enhance respiration and net metabolism or organic matter in the bay. These results suggest that the abundance and respiration of macroaggregates throughout the water column may be responsible for the undersaturation of dissolved oxygen observed in other studies in the BTS. The vertical fluxes of macroaggregates may also contribute to benthic respiration rates. The influence of macroaggregate disaggregation on the operational definition of dissolved and particulate phases should be considered and deserves further study.