Seasonal Dynamics of Microalgal Biomass and Its Biomethanation Potential: A Case Study from the Bay of Gdansk, Poland

Abstract

This study aimed to evaluate the effect of seasonal dynamics of phytoplankton biomass in the Bay of Gdansk (Southern Baltic Sea, Poland) on its usability for anaerobic digestion. Biomass samples were collected between May and October (2023–2024) for quantitative, taxonomic, and chemical analyses as well as for anaerobic digestion in mesophilic periodical bioreactors. Study results demonstrated substantial seasonal variations in the taxonomic composition of phytoplankton, with green algae and dinoflagellates prevailing in the spring, cyanobacteria in the summer, and diatoms in the autumn. These fluctuations were also reflected in the chemical composition of the biomass and its anaerobic digestion efficiency. The highest methane yield of 270 ± 13 mL CH₄/g VS and its highest production rate reaching 32.5 ± 1.6 mL CH₄/g VS·d were recorded in August, i.e., in the period of cyanobacteria predominance with the maximal contents of TOC (51.4 ± 2.1% TS), sugars (599 ± 42 mg/g TS), and lipids (126 ± 13 mg/g TS) in the biomass. In contrast, the lowest biomethanation efficiency was determined in October under diatom prevalence. A strong correlation was found between taxonomic, structural, and chemical properties of the substrate, and anaerobic digestion efficiency. This study’s findings underscore the enormous potential of phytoplankton biomass from summer blooms for energy production as a crucial element of sustainable management of coastal ecosystems and the circular economy.

1. Introduction

Blooms of microalgae and cyanobacteria pose a growing ecological problem worldwide [1,2]. When coupled with climate change, eutrophication, and anthropogenic activities, they lead to various adverse environmental consequences, including oxygen deficit, mass fish kills, deterioration of water quality, and the production of toxins that pose risks to human and animal health [3]. The last decade has brought a tangible increase in the frequency and intensity of blooms, both in fresh and sea waters, especially in strongly urbanized and agricultural areas [4].

The problem of blooms has been particularly severe in the Baltic Sea regions, including specifically the Bay of Gdansk, and is mainly driven by poor water circulation, a relatively shallow bathymetric profile, high inputs of nitrogen and phosphorus from local sources and river tributaries, as well as the specific trophic structure of this ecosystem [5]. Recurrent seasonal blooms not only deteriorate water quality but also strongly affect the functions of local ecosystems, leading to biodiversity loss and undermining the economic use of coastal resources, particularly in recreation, tourism, and fisheries [6].

The increasing severity of algal blooms underscores the necessity of implementing comprehensive and sustainable measures that both mitigate their adverse impacts and facilitate the use of surplus phytoplankton within a circular economy framework. One of the most promising concepts in this respect involves the active removal of biomass from aquatic systems and its subsequent use for bioenergy production [7]. This strategy yields both ecological and economic advantages. Reducing biomass in the water limits its decomposition, improves aerobic conditions, and decreases internal nutrient loading [8]. At the same time, the harvested biomass can be converted into biogas or methane (CH₄) via anaerobic digestion, providing energy and economic benefits [9]. Globally, attempts have already been made to implement such solutions, particularly in regions heavily affected by eutrophication [10]. For example, in China, France, and Denmark, experimental and semi-industrial facilities aimed to convert microalgal biomass into biogas, achieving methane yields ranging from 150 mL CH₄/g VS to as high as 350 mL CH₄/g VS, depending on the biomass taxonomic composition and pretreatment methods applied [11]. The authors of this manuscript also conducted experiments in a semi-technical scale installation, in which phytoplankton biomass was mechanically harvested from the waters of the Vistula Lagoon. Their results indicated that anaerobic digestion of the harvested microalgal biomass produced biogas yields from 244 L/kg VS to 395 L/kg VS, with methane content of the gaseous mixture ranging from 41.4% to 61.9% [12].

In contrast to tropical and subtropical regions, temperate zone ecosystems—such as the southern Baltic Sea—are characterized by pronounced seasonal variability in primary productivity and in the taxonomic composition of phytoplankton assemblages throughout the growing season [13]. These changes directly affect the quality and chemical composition of biomass, including the C/N ratio, the content of structural compounds, and the presence of inhibitory substances, all of which may significantly influence the yield and kinetics of methane fermentation [14]. In the context of biogas plant operation, this translates into challenges of substrate instability, requiring either adjustments of technological parameters in response to variable feedstock or co-digestion with other organic substrates [15].

Despite the growing global interest in phytoplankton-to-bioenergy pathways, no studies to date have addressed these aspects in the Bay of Gdansk, Poland. Data is lacking on seasonal quantitative and qualitative variability of its microalgal biomass, particularly in terms of its utility for biogas production. This represents a critical knowledge gap, especially given the escalating eutrophication pressure and the urgent need for implementing strategies following sustainable development principles.

In view of the above, the present study aimed to provide a comprehensive assessment of the seasonal variability in taxonomic, quantitative, and chemical composition of phytoplankton biomass in the Bay of Gdansk between May and October, with emphasis on its methane and biogas yield potential as well as anaerobic digestion kinetics. The findings constitute the first dataset of this water body and provide a foundation for future research on the practical exploitation of microalgal biomass in renewable energy systems and water quality improvement strategies.

2. Materials and Methods

2.1. Concept of Research Works

The experiment was divided into two stages (S). In the first stage (S1), phytoplankton (microalgal) biomass was collected from the Bay of Gdansk, Poland. The material was subjected to quantitative, taxonomic, and chemical analyses, and its suitability as a substrate for anaerobic digestion was evaluated. In parallel, basic physicochemical parameters of the bay water were also determined. The second stage of the experiment (S2) involved measurements aimed to assess the efficiency of methane fermentation in periodic respirometric bioreactors based on analyses of the volume and qualitative composition of the biogas produced and determinations of the basic indicators of anaerobic digestion kinetics. This stage (S2) was divided into six experimental variants (V), based on the month of the growing season in which the microalgal biomass was collected from the Bay of Gdansk. And thus, six variants corresponding to the successive months of the growing season between May and October (2023–2024) were denoted as V1—May, V2—June, V3—July, V4—August, V5—September, and V6—October. Due to the low phytoplankton biomass concentrations in the waters of the Gdansk Bay in the winter season, the period between November and April was excluded from the study.

2.2. Study Area

The research was conducted in the Bay of Gdansk, a southern embayment of the Baltic Sea located along the Polish coastline. The bay covers approximately 500 km² and has an average depth of 50–60 m, with a maximum depth exceeding 100 m in its central part. The shoreline is highly diversified, encompassing sandy beaches, eroding cliffs, and deltaic formations at river estuaries. The Vistula River, the largest river discharging into the Baltic Sea, strongly influences the hydrology and biogeochemistry of the bay, supplying significant amounts of suspended solids, nutrients, and organic matter.

From a geological and geomorphological perspective, the Bay of Gdansk is a young postglacial formation shaped during the Holocene transgression. Its seabed is characterized by sandy and muddy sediments with locally occurring glacial deposits and morainic structures. Coastal dynamics are driven by both marine and fluvial processes, resulting in sediment accumulation near river mouths and erosion along exposed shorelines. The inner part of the bay (including the Puck Bay) is shallower, partially enclosed, and ecologically distinct, with a more pronounced influence of freshwater inflow and reduced water exchange with the open Baltic.

Climatically, the Bay of Gdansk is situated in the transitional zone between the oceanic and continental climate of Central Europe. The region is characterized by moderate temperatures, with an annual average of 7–8 °C. Winters are relatively mild, with mean January temperatures around −1 to 0 °C, while summers are moderately warm, with mean July values of 17–18 °C. The annual precipitation ranges between 550 and 650 mm, distributed fairly evenly throughout the year. Ice cover may form during severe winters, especially in the shallow inner part of the bay, significantly affecting hydrodynamics and biological processes.

Hydrological conditions are largely controlled by wind-induced currents, freshwater inflows, and stratification patterns typical of the Baltic Sea. Seasonal stratification develops in late spring and persists until early autumn, influencing nutrient availability, oxygen distribution, and phytoplankton productivity. The morphology and restricted water circulation in the Bay result in long retention times and increased nutrient accumulation, which promotes the intensive growth of phytoplankton (microalgae). High nutrient inputs from the Vistula River contribute to eutrophication and recurrent phytoplankton blooms, particularly of cyanobacteria and green algae, which directly affect the quantity and quality of biomass available for biotechnological and bioenergy applications.

The Bay of Gdansk is classified as a eutrophic coastal water body, influenced by a significant inflow of nutrients, also from the Reda and Płutnica rivers and from agricultural and residential catchments. The Bay is protected under the Natura 2000 protected areas network (PLH220032—The Bay of Gdansk and Coastal Waters), and its ecological status is systematically monitored by national and international institutions, including HELCOM programs.

2.3. Microalgal Biomass

The biomass of phytoplankton/microalgae was harvested from the waters of the Bay of Gdansk twice a month between May and October, using plankton nets with a mesh size of 30.0 μm. The harvested biomass was concentrated by means of a laboratory centrifuge (MPW-251, MPW Med. Instruments, Warsaw, Poland). Each experimental variant of the studies addressing anaerobic digestion was conducted with ca. 2.0 L of the biomass concentrated to min. 20 g TS/L. The microalgal biomass sampling point was located at the reference point designated as part of the environmental monitoring conducted by the Institute of Meteorology and Water Management (IMGW), the Helsinki Commission—Baltic Marine Environment Protection Commission (HELCOM), and the Chief Inspectorate of Environmental Protection (GIOŚ). The point is located near Sopot, Poland (designation: GDY SOP, 54°40′17″ N, 18°56′80″ E), approximately 2 km away from the beach in Sopot, at the pier’s height, within the range of shallow waters of the Bay of Gdansk, which extend into the deeper reaches of the Bay of Gdansk. The approximate location of the phytoplankton sampling point is shown in Figure 1.

2.4. Inoculum of Anaerobic Respirometric Digesters

The inoculum for anaerobic digestion studies was sourced from the anaerobic digesters of the Municipal Wastewater Treatment Plant “Łyna” in Olsztyn, Poland (53°48′49″ N, 20°27′1″ E). The facility operates under mesophilic conditions (37 ± 1 °C), with the hydraulic retention time (HRT) of 20–30 days, and an average organic load rate (OLR) of 2.0–3.0 kg VS/m³·d. Characteristics of the anaerobic sludge is as follows (mean values ± standard deviations, n = 5): total solids (TS): 3.5 ± 0.5%, volatile solids (VSs): 65.0 ± 4.0% TS, total organic carbon (TOC): 320 ± 25 mg/g TS, total carbon (TC): 370 ± 20 mg/g TS, total nitrogen (TN): 25 ± 3 mg/g TS, total phosphorus (TP): 6.3 ± 1.0 mg/g TS, C/N ratio: 12.8 ± 0.5, pH 7.3 ± 0.1, and redox potential (RP): −250 ± 20 mV.

2.5. Respirometric Measurements of Biogas Production

Biogas production efficiency and biogas qualitative composition, depending on the microalgal biomass composition, were measured in the subsequent months of the growing season using OxiTop^® Control respirometric systems (WTW, Troisdorf, Germany). Experiments were conducted under controlled conditions in a thermostatic cabinet maintaining a constant fermentation temperature of 38 ± 1.0 °C. The total capacity of each bioreactor was 500 mL, with 200 mL of the working volume consisting of anaerobic sludge serving as the inoculum. The appropriate amount of microalgal biomass was fed into the inoculum, achieving an initial organic load rate (OLR) of 5.0 g VS/L. The remaining space in the bioreactor was the gas phase, enabling the accumulation and measurement of the generated biogas. Before the exact digestion began, the inoculum was pre-incubated at 38 ± 1.0 °C for 10 days, without substrate addition, in order to minimize the influence of residual, easily accessible organic matter contained in the anaerobic sludge, which allowed for a more precise identification of fermentation effects with solely the tested substrate.

Once the bioreactors had been fed with the inoculum and the organic substrate, their contents were flushed with technical nitrogen (flushing time—2 min, flow rate—100 L/h) to eliminate oxygen and ensure anaerobic conditions. The hydraulic retention time (HRT) in the digesters was set at 24 days, ensuring sufficient time for the methanogenesis process to occur. Fermentation was considered complete when the daily increase in biogas volume over three consecutive days did not exceed 1%. The OxiTop^® Control system used in the study enabled recording the partial pressure of the generated gas, ensuring tightness, mixing of the contents, and collecting samples for qualitative analyses (GC analysis). A control sample containing only the inoculum was run simultaneously, allowing for the correction of the results using the methanogenic activity of the sludge.

The volume and molar amount of biogas produced were calculated based on the ideal gas law, according to the equation:

n = p·V/R·T

where n—number of moles of biogas [mol], p—partial pressure of gases in the reactor [Pa], V—volume of the gas phase in the reactor [m³], R—gas constant (8.314 J/mol·K), and T—absolute temperature of fermentation [K].

Based on the number of moles of biogas, its volume was determined under standard conditions (T = 273 K, p = 1013 hPa) according to the Avogadro equation:

V = n·V_mol

where V—volume of biogas [L], n—number of moles [mol], and V_mol—molar volume of biogas under standard conditions (22.4 L/mol).

The modified Gompertz model was applied in order to determine the kinetics of anaerobic digestion and to quantify the potential and dynamic behavior of biogas and methane production. It is widely used to describe cumulative gas production in batch systems, particularly for microbial processes with lag and exponential phases. The modified Gompertz model is defined by the following equation:

$$V(t)=V_{max}·exp\{-exp\left[{\displaystyle \frac{R_{max·e}}{V_{max}}}·\left(\lambda -t\right)+1\right]\}$$

where

-

V(t) is the cumulative volume of biogas or methane produced at time t [mL/g VS],

-

V_max is the maximum (asymptotic) biogas/methane yield [mL/g VS],

-

R_max is the maximum production rate [mL/g VS·d],

-

λ is the lag phase duration [d],

-

t is the fermentation time [d],

-

e is Euler’s constant (≈2.71828).

It has a sigmoidal shape and enables accurate estimation of the three key phases of the digestion process: initial microbial adaptation (λ), the exponential gas production phase (defined by R_max), and the saturation plateau (V_max). The model parameters V_max, R_max, and λ were estimated by non-linear regression of the experimental cumulative biogas and methane data using least-squares fitting methods. Curve fitting was performed using computational tools such as MATLAB R2023a (nlinfit), Python 3.9.0 (SciPy—curve_fit), or R 4.3.0 (nls), depending on the computational environment. Smoothing of the raw data was applied prior to the fitting to minimize the influence of experimental noise. The goodness of fit was evaluated using the coefficient of determination (R²) and the root-mean-square error (RMSE). Parameters were estimated separately for both total biogas and methane production curves to enable comparative kinetic analysis between seasonal biomass batches. The non-linear regression analysis was performed to determine the fermentation kinetics and reaction rate constants (k), using the STATISTICA 13.3 PL package (StatSoft, Inc., Tulsa, OK, USA) by fitting the models to the experimental biogas production curves.

2.6. Analytical Methods

Determinations of phytoplankton were conducted using an inverted microscope Axiovert 35 (Zeiss, Oberkochen, Germany) following recommendations adopted by HELCOM [16]. Phytoplankton biomass was estimated based on standard cell volumes, and results were expressed in µgC/L using formulas consistent with the latest HELCOM recommendations. To enable comparability of data on phytoplankton biomass concentration, concentrations given in µgC/L were converted to total solids in mg TS/m³. The conversion methodology was based on current guidelines [16] and recommendations [17]. The adopted content of organic carbon in the dry matter of phytoplankton determined for the investigated taxonomic groups and the µgC to µgTS conversion factors were as follows: green algae (Chlorophyta)—0.48 and 2.08; diatoms (Bacillariophyta)—0.40 and 2.50; cyanobacteria (Cyanobacteria)—0.43 and 2.33; and dinoflagellates (Dinophyta)—0.50 and 2.00, respectively. In addition, the TS concentration of phytoplankton biomass was determined directly by drying at 60 °C to a constant weight. The percentages of individual taxonomic components in the biomass were estimated. Analyses were performed in accordance with the HELCOM methodology, i.e., Annex C-4 (chlorophyll-a) and C-6 (phytoplankton) [16]. A Unico 2800 UV-VIS spectrophotometer (HITACHI, Ibaraki, Japan) was used to determine chlorophyll-a content. Water temperature and salinity measurements were performed according to HELCOM (1) ANNEX C-2 and B-8 guidelines using a CTD Ocean Seven 304 probe (IDRONAUT, Brugherio, Italy) or a CTD SBE49 probe (Sea-Bird Scientific, Bellevue, WA, USA). Water transparency measurements were performed according to HELCOM ANNEX C-2 methodology using a Secchi disk [16].

The VS analysis was performed gravimetrically by incinerating the biomass sample at 550 °C in a muffle furnace (LAC L model, Dąbrowica, Poland) and then weighing the remaining ash (DanLab AX423 analytical scale, Białystok, Poland). Dried biomass samples (105 °C, 24 h) were analyzed for contents of total carbon (TC), total organic carbon (TOC), and total nitrogen (TN) using a Flash 2000 elemental analyzer (Thermo Scientific, Delft, The Netherlands). Protein content was calculated using a nitrogen-to-protein conversion factor of 6.25. Carbohydrate content was determined spectrophotometrically at 600 nm using the anthrone method with glucose as a standard (DR 2800, Hach-Lange GmbH, Düsseldorf, Germany). Lipid content of the dried biomass was determined by means of the Soxhlet extraction using hexane as the solvent (Büchi, Flawil, Switzerland). Prior to the extraction, the samples were ultrasound-treated for 30 s using a UP400S processor (Hielscher Ultrasonics, Teltow, Germany) set to 100% amplitude. Biogas was extracted from qualitative respirometers using gas-tight syringes and analyzed chromatographically on an Agilent 7890 A GC apparatus (Agilent Technologies, Santa Clara, CA, USA).

2.7. Statistical Methods

The experiment was conducted in four replications. Statistical analysis of data was performed using Statistica 13.3 package (StatSoft Inc., Tulsa, OK, USA), at a significance level of α = 0.05. Prior to the analysis of differences between groups, normality of data distribution was evaluated using the Shapiro–Wilk’s test, whereas homogeneity of variance within comparisons was checked with Levene’s test. One-way analysis of variance (ANOVA) was deployed to evaluate differences between mean values, and honestly significant difference (HSD) Tukey’s test—to identify significant differences between individual groups.

3. Results and Discussion

3.1. Water Quality Parameters

The analyzed growing season exhibited pronounced changes in the physicochemical properties of water from the Bay of Gdansk, which in turn significantly affected the planktonic ecosystem dynamics (

Table 1. Basic parameters of water sampled from the Bay of Gdansk in the subsequent months of the growing season.

Parameter	Unit	Month
		May	June	July	August	September	October
Salinity	[PSU]	6.5 ± 0.7	7.0 ± 0.2	7.5 ± 0.3	7.8 ± 0.6	7.6 ± 0.4	6.9 ± 0.5
Temperature	[°C]	10 ± 4.2	12 ± 2.2	15 ± 0.8	17 ± 2.8	15 ± 0.7	12 ± 2.3
Oxygen	[mg/L]	8.2 ± 0.5	7.8 ± 0.1	7.5 ± 0.2	7.3 ± 0.4	7.6 ± 0.1	8.0 ± 0.3
Transparency	[m]	4.5 ± 0.3	4.2 ± 0.1	4.0 ± 0.2	3.8 ± 0.4	4.1 ± 0.1	4.3 ± 0.2
Chlorophyll-a	[µg/L]	7.1 ± 0.6	10.5 ± 1.2	25.0 ± 1.3	31.4 ± 2.8	12.9 ± 1.5	6.2 ± 0.5
Feopigments	[µg/L]	1.54 ± 0.23	2.62 ± 0.11	3.71 ± 0.09	4.83 ± 0.15	3.77 ± 0.12	1.69 ± 0.07

).

Water temperature was observed to increase successively from 10.0 ± 4.2 °C in May to the maximal value of 17.0 ± 2.8 °C recorded in August, and then to decrease to 12.0 ± 2.3 °C in October (Table 1). This water temperature increase was accompanied by a successive reduction in water transparency, i.e., from 4.5 ± 0.3 m in May to the lowest value of 3.8 ± 0.4 m recorded in August, which points to the enhanced development of suspended matter and biomass in the summer period [18]. This trend reversed in the autumn, when water transparency increased to 4.3 ± 0.2 m in October, resulting in a decline in primary production. The temperature of water in the Baltic Sea has been observed to increase within the last 100 years and is estimated to rise by another 1.1–3.2 °C by the end of this century [19]. Higher temperatures intensify water stratification, thereby promoting summer cyanobacterial blooms and increasing their impact on fisheries and the recreational use of Baltic Sea coastal waters [20].

Oxygen concentration in water exhibited minor fluctuations, remaining relatively high, from 8.2 ± 0.5 mg O₂/L in May to the lowest value of 7.3 ± 0.4 mg O₂/L in August, and afterwards increased to 8.0 ± 0.3 mg O₂/L in October (Table 1). The reduction in oxygen content of water in the summer months is probably due to increased oxygen consumption for organic matter degradation and intensive respiration processes of microorganisms that accompany the peak of phytoplankton biomass production [21]. Long-term studies of oxygen concentration in the Bay of Gdansk have indicated that the mean concentration of dissolved oxygen was at 12 mg O₂/L, with two peaks recorded in March/April and November [22]. This also applies to the inner part of the Bay of Gdansk, which is well-oxygenated, although partially separated from the circulation of the open sea by the Hel Peninsula [23]. However, with climate warming and the increasing likelihood of seasonal severe thermal stratification, periodic oxygen deficits may develop in the coastal zone of the Bay of Gdansk in the future, similar to those observed in the Stockholm Archipelago and the Finnish Archipelago Sea [24].

Concentrations of chlorophyll-a, as an indicator of phytoplankton biomass, increased significantly from 7.1 ± 0.6 µg/L in May, peaked to 31.4 ± 2.8 µg/L in August, to finally dropped to 6.2 ± 0.5 µg/L in October (Table 1). These increases in chlorophyll-a concentration correlated with the observed reduction in water transparency and a feopigment concentration increase from 1.54 ± 0.23 µg/L in May to 4.83 ± 0.15 µg/L in August, pointing to the enhanced degradation of phytoplankton pigments and organic matter circulation (Table 1). Statistical analysis of data demonstrated significant (p ≤ 0.05) differences in the values of these parameters between the spring and summer seasons, thereby confirming seasonal intensification of biomass production and degradation processes. Variability of water salinity was relatively low, ranging from 6.5 ± 0.7 PSU in May to 7.8 ± 0.6 PSU in August, with a downward tendency observed in the later period of the growing season, i.e., to 6.9 ± 0.5 PSU in October (Table 1). These fluctuations could be due to the impacts of hydrological factors, like runoff of fresh water and the mixing of water strata, which also affect the availability of nutrients and environmental conditions developed for the phytoplankton [25]. In addition, seasonal climate variability strongly influences phytoplankton growth, while the peak algal growth observed in the summer is also determined by the amount of solar radiation reaching their chloroplasts [18].

The increase in water temperature coupled with its reduced transparency promote the intensive development of phytoplankton biomass, particularly cyanobacteria, which are able to function in conditions of limited light and high temperatures [26]. In the summer period, when the contents of chlorophyll-a and feopigments peaked, water transparency decreased, which is a typical phenomenon indicative of the enhanced degradation of dead organic matter and suspended matter, which further diminishes light penetration [27]. In turn, water temperature decreased and water transparency increased, as observed in the fall promoted the predominance of organisms preferring cooler and better-lit environments, which was also reflected in reduced biomass production. Literature data unequivocally demonstrate that cyanobacterial blooms in the Baltic Sea are a recurrent summer phenomenon, often covering extensive areas of the basin [28]. Following the spring diatom bloom, nitrogen concentrations are substantially depleted, while excess dissolved phosphorus remains available during the summer months. This nutrient imbalance creates favorable conditions for cyanobacteria, which are able to fix atmospheric nitrogen under nitrogen-limiting conditions [29]. In addition, high solar radiation combined with a low wind speed reduces vertical mixing of waters, enhances surface-layer stratification, and elevates water temperature, thereby promoting the initiation and proliferation of cyanobacterial blooms [28].

3.2. Quantitative and Taxonomic Characteristics of the Biomass

During the analyzed growing season, pronounced shifts were observed in both the taxonomic composition of phytoplankton and the total biomass and chlorophyll-a concentrations in the waters of the Bay of Gdansk. These changes followed a clear seasonal gradient, consistent with the classical model of plankton succession in estuarine ecosystems of the temperate clime zone: starting with the dominance of spring assemblages, progressing to summer cyanobacterial blooms, and culminating in autumn diatom communities [30].

Table 2. Taxonomic characteristics and concentration of microalgal biomass in waters of the Bay of Gdansk in the analyzed months of the growing season.

Month	Prevailing Groups and Species and Their Contribution in TS	Chlorophyll-a Content and Biomass Concentration
May	- Gymnodinium spp. (dinoflagellates) (43% TS) - Chlamydomonas spp., Monoraphidium spp. (green algae) (52% TS) - Skeletonema costatum (diatoms) (5% TS)	Chlorophyll-a: 7.1 ± 0.6 µg/L Biomass: 270 ± 31 mg TS/m3
June	- Dinophysis acuminata, Prorocentrum micans (dinoflagellates) (41% TS) - Chlorella spp., Scenedesmus spp. (green algae) (42% TS) - sporadically: Aphanizomenon spp. (cyanobacteria) (17% TS)	Chl-a: 10.5 ± 1.2 µg/L Biomass: 430 ± 39 mg TS/m3
July	- Nodularia spumigena, Aphanizomenon flos-aquae, Dolichospermum spp. (cyanobacteria) (84% TS) - Ceratium hirundinella (dinoflagellates) (9% TS) - Chlorella spp., Scenedesmus spp. (green algae) (7% TS)	Chl-a: 25.0 ± 1.3 µg/L Biomass: 1130 ± 84 mg TS/m3
August	- Nodularia, Dolichospermum, Microcystis aeruginosa (cyanobacteria) (81% TS) - Ceratium spp., Protoperidinium spp. (dinoflagellates) (12% TS) - Chlorella spp., Scenedesmus spp. (green algae) (4% TS) - Skeletonema costatum (diatoms) (3% TS)	Chl-a: 31.4 ± 2.8 µg/L Biomass: 1250 ± 107 mg TS/m3
September	- Nodularia spumigena, Aphanizomenon flos-aquae, Dolichospermum spp. (cyanobacteria) (32% TS) - Chaetoceros spp., Coscinodiscus spp. (diatoms) (39% TS) - Peridinium spp. (dinoflagellates) (29% TS)	Chl-a: 12.9 ± 1.5 µg/L Biomass: 580 ± 64 mg TS/m3
October	- Skeletonema marinoi, Thalassiosira spp. (diatoms) (76% TS) - Nodularia spumigena, Aphanizomenon flos-aquae, Dolichospermum spp. (cyanobacteria) (13% TS) - Peridinium spp. (dinoflagellates) (11% TS)	Chl-a: 6.2 ± 0.5 µg/L Biomass: 340 ± 21 mg TS/m3

presents data on the observed changes in the taxonomic and quantitative structure of the phytoplankton biomass.

In the springtime, i.e., in May and June, mainly cyanobacteria and dinoflagellates were observed to predominate (Table 2). In May, green algae accounted for the largest share in the total dry matter of phytoplankton (52% TS), followed by dinoflagellates (43% TS). The population of diatoms was small (5% TS), whereas cyanobacteria were not detected. In June, the taxonomic structure of the biomass was more balanced, with approximately 40% TS shares of green algae and dinoflagellates, and cyanobacteria appearing at ca. 17% TS. The increase in cyanobacteria abundance observed in June may indicate an intensification of eutrophication processes, reinforced by rising water temperatures and the persistence of a stable water column, which collectively create favorable conditions for the development of nitrogen-fixing taxa [31]. In this period, chlorophyll-a content increased from 7.1 ± 0.6 µg/L to 10.5 ± 1.2 µg/L, and phytoplankton biomass concentration from 270 ± 31 mg TS/m³ to 430 ± 39 mg TS/m³, which was indicative of the successive enhancement of photosynthetic activity and primary production (Table 2). According to the available literature, spring blooms in the Baltic Sea are typically composed of diatoms and dinoflagellates, which together constitute the bulk of the bloom biomass [32]. However, recent studies have revealed considerable variability in the contribution of these taxa in the spring blooms. Still, the factors and mechanisms driving the dominance of cold-water dinoflagellates in spring phytoplankton communities remain poorly understood, likewise their pronounced prevalence in the ice-free central Baltic Sea [20]. Findings reported by Camarena-Gómez et al. [33] further suggest that an increasing share of dinoflagellates during spring blooms may, in the coming years, exert a strong influence on the structure and functioning of associated bacterial communities, which represent the primary consumers of phytoplankton-derived dissolved organic matter, thereby affecting pelagic mineralization of organic matter.

In the summer months (July and August), a pronounced shift was observed in both the qualitative and quantitative structure of phytoplankton biomass. During this period, phytoplankton was predominated by cyanobacteria, which accounted for 84% of biomass dry matter (TS) in July and 81% TS in August (Table 2). Other groups, such as dinoflagellates and green algae, played only a minor role in this period, accounting for only 9% TS and 7% TS in July, and 12% TS and 4% TS in August, respectively. Notably, August samples also revealed the presence of colonial forms of Microcystis aeruginosa, a species widely recognized as potentially toxic. According to the literature data, non-nitrogen-fixing algal genera, such as Microcystis, require the presence of nitrates in the water, which can be supplied in excess through surface runoff from agricultural areas [34]. Conversely, phosphorus excess in nitrogen-depleted aquatic ecosystems promotes the development of nitrogen-fixing taxa [34]. This stage of phytoplankton succession corresponds to the typical summer cyanobacterial bloom, observed under conditions of elevated temperature, a stable water column, and reduced competition from other phytoplankton groups [20]. Furthermore, Beltran-Perez and Waniek [35] emphasized that it is the energetic yield derived from a stratified water column that substantially increases bloom intensity by reinforcing vertical stratification. In the present study, the taxonomic dominance of cyanobacteria also resulted in the highest chlorophyll-a concentrations, ranging from 25.0 ± 1.3 µg/L in July to 31.4 ± 2.8 µg/L in August, and an increase in phytoplankton biomass concentration reaching 1130 ± 84–1250 ± 107 mg TS/m³ (Table 2). Such values may indicate that the thresholds defining good ecological status under the Water Framework Directive have been surpassed [36].

September and October were the months of successive reorganization of the structure of phytoplankton community, pointing to the onset of autumn succession. The contribution of cyanobacteria decreased then to 32% TS in September and 13% TS in October (Table 2), in favor of diatoms, which accounted for 39% TS and as much as 76% TS, respectively. The increasing importance of diatoms, such as Chaetoceros spp., Coscinodiscus spp., Skeletonema marinoi, and Thalassiosira spp., is due to water temperature increase, increased intensity of vertical stratification, and improvement in lighting conditions upon reduced bloom [37]. According to Hjerne et al. [38], the strong wind-driven mixing of the water strata promotes the redistribution of diatom resting stages and nutrients throughout the water column. Dinoflagellates persisted throughout the growing season, contributing notably in September (29% TS); however, they never dominated at any point during the season. The decreasing content of chlorophyll-a, from 12.9 ± 1.5 µg/L in September to 6.2 ± 0.5 µg/L in October, coupled with biomass concentration reduction from 580 ± 64 mg TS/m³ to 340 ± 21 mg TS/m³, suggest substantial suppression in the photosynthetic activity in the terminal period of the growing season (Table 2).

Analysis of the taxonomic structure throughout the study period revealed that the most pronounced qualitative shifts occurred between June and July, when communities dominated by green algae and dinoflagellates were abruptly replaced by cyanobacteria. This transition was most likely driven by a combination of abiotic factors, i.e., increased temperature, reduced mixing intensity, and limited availability of inorganic nitrogen, all of which promote the taxa capable of atmospheric nitrogen fixation [28]. Apart from photoautotrophy, cyanobacteria employ a range of ecophysiological strategies (e.g., buoyancy regulation through aggregates of gas vesicles, rapid cell division, and the capacity to accumulate phosphorus and trace metals), enabling them to outcompete eukaryotic algae and establish mass dominance within phytoplankton assemblages [39]. By contrast, the transition to diatom-dominated communities in the autumn can be interpreted as a return to a more balanced ecological state, predominated by taxa with short life cycles and high photosynthetic efficiency [37]. These shifts reflect dynamic ecological processes in the waters of the Baltic coastal zone and may serve as valuable indicators for assessing the impacts of climate change and anthropogenic pressures on the aquatic ecosystem status.

3.3. Chemical Composition of Microalgal Biomass

During the growing season, pronounced and statistically significant changes were observed in the chemical composition of phytoplankton biomass, expressed in the contents of key nutrients and major biochemical fractions. These differences were closely linked to the seasonal taxonomic succession and reflected the dynamic ecological processes in the estuarine waters of the Gdansk Bay. Their analysis provides deeper insight into the mechanisms driving phytoplankton productivity and biochemical quality, which are of critical importance both for ecosystem ecology and potential biotechnological applications.

Table 3. Chemical composition of phytoplankton biomass harvested from the Bay of Gdansk in the subsequent months of the growing season.

Parameter	Unit	Month
		May	June	July	August	September	October
TOC	[% TS]	43.5 ± 1.5 a	45.6 ± 1.7 a	50.1 ± 2.0 b	51.4 ± 2.1 b	47.5 ± 1.8 ab	44.5 ± 1.6 a
TN	[% TS]	6.5 ± 1.1 ab	7.2 ± 0.9 b	6.0 ± 0.3 a	5.5 ± 0.9 a	6.3 ± 0.4 ab	7.1 ± 0.6 b
TP	[% TS]	1.21 ± 0.11 ab	1.35 ± 0.15 b	1.00 ± 0.12 a	0.83 ± 0.10 a	1.15 ± 0.14 ab	1.32 ± 0.15 b
C/N	-	6.7 ± 0.3 a	6.3 ± 0.3 a	10.0 ± 0.5 b	11.4 ± 0.6 c	7.5 ± 0.4 ab	6.3 ± 0.3 a
VS	[%]	77.7 ± 1.5 ab	79.0 ± 1.7 b	82.2 ± 2.0 c	83.5 ± 2.1 c	77.3 ± 1.8 ab	75.0 ± 1.6 a
Protein	[mg/g TS]	406 ± 31 b	438 ± 38 b	313 ± 37 a	281 ± 40 a	375 ± 32 ab	438 ± 44 b
Lipids	[mg/g TS]	91 ± 9 a	105 ± 11 ab	122 ± 12 b	126 ± 13 b	95 ± 10 a	83 ± 9 a
Sugars	[mg/g TS]	504 ± 33 ab	462 ± 36 a	567 ± 39 c	599 ± 42 c	530 ± 36 bc	477 ± 32 ab

Different superscript letters (a, b, c, ab, bc) within the same row indicate statistically significant differences between the analyzed variables. The same letters indicate no significant difference.

presents the chemical composition of phytoplankton biomass.

The TOC content showed an upward trend between May and August, when it increased from 43.5 ± 1.5% TS to 51.4 ± 2.1% TS, respectively (Table 3). In September and October, its values were significantly (p ≤ 0.05) lower, reaching 47.5 ± 1.8% TS and 44.5 ± 1.6% TS, respectively. The observed trend was strongly correlated with VS content, which increased in the analogous periods from 77.7 ± 1.5% TS in May to the maximal value of 83.5 ± 2.1% TS recorded in August, and afterwards decreased to 77.3 ± 1.8% TS in September and 75.0 ± 1.6% TS in October (Table 3). The high TOC and VS contents determined in the summer months point to increased accumulation of organic matter, mainly due to the development of cyanobacteria whose cells are rich in organic matter and poor in minerals [40]. In turn, decreases noted in their contents in the autumn period are consistent with the shift to phytoplankton communities predominated by diatoms, whose siliceous frustules boost the inorganic fraction of the biomass [41].

The TN content varied across the growing season from the maximal values of 7.2 ± 0.9% TS recorded in June and 7.1 ± 0.6% TS in October to the minimal value of 5.5 ± 0.9% TS determined in August (Table 3); however, the changes in its concentration in the biomass were statistically insignificant (p ≤ 0.05). A decrease in the mean TN concentrations in the biomass noted in the summer months may be due to the limited availability of nitrogen in the water column during the peak primary production [42]. This period was also characterized by the predominance of diazotrophic cyanobacteria capable of fixing atmospheric nitrogen but synthesizing biomass with a low content of enzymatic proteins. In aquatic ecosystems with limited nitrogen availability, atmospheric nitrogen fixation provides diazotrophic cyanobacteria with a competitive advantage over non-diazotrophic taxa [43]. Throughout the growing season, the C/N ratio ranged from 6.3 ± 0.3 (June and October) to 11.4 ± 0.6 (August) (Table 3). Its pronounced increase in the summer reflects a strong nitrogen deficit relative to carbon in microalgal cellular composition, indicating potential nitrogen stress within the planktonic community [44]. According to Chai et al. [42], the coupling of nitrogen and phosphorus cycling in bottom sediments under low-oxygen conditions can effectively alter the form and stoichiometric ratio of nitrogen to phosphorus in the water column, thereby regulating the dynamics of algal blooms. Low nitrogen concentrations compared to dissolved phosphorus result primarily from the high intensity of denitrification in Baltic Sea sediments, which reduces nitrates and nitrites to gaseous products: nitric oxide, nitrous oxide, and dinitrogen, thereby contributing to a lowered N/P ratio in pelagic waters and promoting the development of diazotrophic cyanobacteria [45]. The protein content of the phytoplankton biomass was directly correlated with total nitrogen levels, reaching maximum values in June (438 ± 38 mg/g TS) and October (438 ± 44 mg/g TS), and the minimum in August (381 ± 40 mg/g TS) (Table 3). These findings indicate that during summer, phytoplankton in the Bay of Gdansk exhibited deteriorated trophic quality, typical of cyanobacterial blooms [46].

The lipid fraction increased in the biomass dry matter since May (91 ± 9 mg/g TS), through June (105 ± 11 mg/g TS), and peaked in July (122 ± 12 mg/g TS) and August (126 ± 13 mg/g TS), (Table 3). Afterwards, this fraction of phytoplankton biomass decreased to 95 ± 10 mg/g TS in September to ultimately reach the minimum value of 83 ± 9 mg/g TS in October. The lipid content increase observed in the summer season can be attributed to microalgal responses to nitrogen stress and high light availability, namely conditions that promote the accumulation of storage lipids in the form of triacylglycerols, as well as to the synthesis of membrane lipids and buoyancy structures (e.g., gas vesicles in cyanobacteria) [47,48,49]. Temperature, salinity, and light intensity have been identified as primary stressors that enhance lipid accumulation in microalgal cells [50]. Lipids also play a crucial role in determining algal dominance and succession, as certain dissolved polyunsaturated fatty acids can cause damage to plasma membranes, accounting for their potential allelopathic effect [51]. The decline in lipid content noted in the autumn coincided with the predominance of diatoms, which generally exhibit lower capacity for lipid synthesis and storage compared to green algae or cyanobacteria [52]. Studies on diatoms have demonstrated that enhanced synthesis of the soluble carbohydrate—chrysolaminarin, driven by overexpression of the phosphoglucomutase gene, can reduce lipid accumulation [53].

The content of carbohydrates increased significantly in the summer months from 504 ± 33 mg/g TS in May to the maximum value of 599 ± 42 mg/g TS in August (Table 3). This increase can be attributed to the over-production of exopolysaccharides (EPS) by mucilaginous cyanobacteria (Dolichospermum spp., Microcystis aeruginosa), which under nitrogen stress conditions intensively synthesize EPS, facilitating the colonization of the euphotic zone [54]. The increase recorded in EPS at low nitrogen concentrations is due to an increased C/N ratio, as under these conditions, carbon bound in the photosynthesis process is mainly consumed by cells for EPS synthesis [55]. Phosphorus excess is also deemed an important factor contributing to the development of mucilage-producing cyanobacteria [56]. An overview of literature indicates that the development of such cyanobacteria as Anabaena spp., Dolichospermum flos-aquae, Lyngbya kuetzingii, and Microcystis aeruginosa was positively correlated with phosphorus content in the medium [57]. In October, despite a relative low TN, the content of sugars was still high (477 ± 32 mg/g TS), presumably due to photosynthetic activity of diatoms and their accumulation of storage carbohydrates (chrysolaminarin) [58].

3.4. Anaerobic Digestion

Seasonal methane fermentation analyses of the phytoplankton biomass harvested from the Bay of Gdansk revealed significant (p ≤ 0.05) differences in both total biogas and methane yields, as well as in the kinetic parameters of the digestion process. These differences were strongly associated with the taxonomic composition of the phytoplankton communities, the chemical composition of the biomass, and the concentrations of chlorophyll-a and total plankton biomass. The parameters characterizing the course and efficiency of the anaerobic digestion of the harvested phytoplankton biomass are summarized in

Table 4. Biogas and methane production yields, and characteristic kinetic parameters of anaerobic digestion of phytoplankton biomass harvested from waters of the Bay of Gdansk in subsequent months of the growing season.

Parameter	Unit	Month
		May	June	July	August	September	October
Biogas yield	mL/g VS	365 ± 12 a	378 ± 12 a	421 ± 17 b	430 ± 15 b	386 ± 12 a	355 ± 13 a
Biogas production rate	mL/g VS·day	47.5 ± 2.0 ab	45.0 ± 2.3 a	54.6 ± 2.5 c	55.9 ± 2.7 c	46.2 ± 1.6 ab	33.6 ± 1.8 d
Biogas production rate constant	1/day	0.13 ± 0.01 a	0.12 ± 0.01 b	0.13 ± 0.02 a	0.13 ± 0.02 a	0.12 ± 0.01 b	0.11 ± 0.01 c
CH4 content	%	56.5 ± 1.7 a	58.1 ± 1.2 ab	62.4 ± 1.9 c	63.0 ± 2.1 c	59.3 ± 1.2 b	56.8 ± 1.7 a
CH4 yield	mL/g VS	206 ± 10 a	218 ± 10 a	262 ± 11 b	270 ± 13 b	229 ± 9 a	201 ± 8 a
Biogas production rate	mL/g VS·day	20.6 ± 1.1 a	23.9 ± 1.3 ab	31.4 ± 1.9 c	32.5 ± 1.6 c	25.3 ± 1.3 b	18.0 ± 1.0 d
Biogas production rate constant	1/day	0.10 ± 0.01 a	0.11 ± 0.01 a	0.12 ± 0.01 a	0.12 ± 0.01 a	0.11 ± 0.01 a	0.11 ± 0.01 a

Different superscript letters (a, b, c, d, ab) within the same row indicate statistically significant differences between the analyzed variables. The same letters indicate no significant difference.

, while the parameters resulting from the use of the Gompertz model are shown in

Table 5. Estimation of modified Gompertz model parameters and model performance metrics for biogas and methane production in subsequent months of the growing season.

Month	Biogas/CH4	Gompertz A (L/kg VS)	Rm (L/kg VS·d)	λ (Days)	R2	RMSE (L/kg VS)
May	Biogas	365	32	1	0.996	6.48
	CH4	206	18	1	0.999	1.57
June	Biogas	365	35	1	0.995	7.71
	CH4	206	19	1	0.998	2.44
July	Biogas	420	40	1	0.991	10.16
	CH4	262	24	1	0.993	5.18
August	Biogas	430	42	1	0.998	4.37
	CH4	270	25	1	0.995	4.21
September	Biogas	383	38	1	0.999	1.65
	CH4	228	20	1	0.992	5.47
October	Biogas	305	28	1	0.997	5.97
	CH4	164	15	1	0.998	2.95

.

In May, the biogas yield reached 365 ± 12 mL/g VS and CH₄ content in the fermentation gas reached 56.5 ± 1.7% (Table 4, Figure 2), resulting in 206 ± 10 mL CH₄/g VS production (Table 4, Figure 2). The maximum rate of CH₄ generation reached 20.6 ± 1.1 mL/g VS·d, and the biogas production rate constant reached k = 0.10 ± 0.01 1/d (Table 4). These indicators reflect moderate reactivity of the substrate, which in this period showed a relatively balanced composition, including TOC (43.5 ± 1.5% TS) and TN (6.5 ± 0.4% TS), which caused a low C/N ratio of 6.7 ± 0.3 and a high content of protein in the biomass, i.e., 406 ± 31 mg/g TS (Table 3). The presence of green algae and dinoflagellates among the predominating phytoplankton taxa (Table 2) is expected to promote high biodegradability of phytoplankton biomass, but the presence of structural fractions could have diminished methane fermentation efficiency [59].

June brought a significant (p ≤ 0.05) increase in biogas yield to 378 ± 12 mL/g VS and CH₄ yield to 218 ± 10 mL/g VS, with CH₄ content of the biogas peaking to 58.1 ± 1.2% (Table 4, Figure 2). The rate of CH₄ production increased significantly (p ≤ 0.05) to 23.9 ± 1.3 mL/g VS·d, and the methanogenesis rate constant reached 0.11 ± 0.01 1/d (Table 4). In July, when the phytoplankton biomass was predominated by cyanobacteria (84% TS) and the chemical composition of the substrate showed the highest TOC level (50.1 ± 2.0% TS), high contents of lipids (122 ± 12 mg/g TS) and sugars (567 ± 39 mg/g TS), and the lowest protein content across the growing season (313 ± 37 mg/g TS), the CH₄ yield increased to 262 ± 11 mL/g VS (Table 3 and Table 4, Figure 2). Biogas production reached 421 ± 17 mL/g VS, and CH₄ content of the fermentation gas was at 62.4 ± 1.9% (Table 4, Figure 2). The kinetic parameters were also high, i.e., the maximum CH₄ production rate was 31.4 ± 1.9 mL/g VS·d, and k = 0.12 ± 0.01 1/d (Table 4). These results reflect the positive impact of high contents of carbohydrates and lipids in the cyanobacterial biomass, which are easily available for microorganisms and highly degradable under anaerobic conditions [60].

August brought further intensification of fermentation, when biogas yield peaked to 430 ± 15 mL/g VS in the growing season, and CH₄ yield reached 270 ± 13 mL/g VS, with the highest CH₄ content of the biomass determined at 63.0 ± 2.1% (Table 4, Figure 2). The CH₄ production rate was also at the peak—32.5 ± 1.6 mL/g VS·d, and the kinetic constant remained high (k = 0.12 ± 0.01 1/d), (Table 4). The biomass harvested in that period had the highest contents of TOC (51.4 ± 2.1% TS) and VS (83.5 ± 2.1% TS), high contents of carbohydrates (599 ± 42 mg/g TS) and lipids (126 ± 13 mg/g TS), as well as the lowest contents of TN (4.5 ± 0.6% TS) and protein (281 ± 40 mg/g TS), (Table 3). The above findings confirm that the mucilaginous cyanobacteria (Microcystis, Dolichospermum) synthesizing polysaccharide fractions (EPS) represented a super-highly fermentable substrate. Cyanobacteria accumulate carbon in various forms, with glycogen and EPS prevailing [61]. According to literature data, the biomass of Microcystis genus algae has a carbohydrate content around 10–30%, lipid content of 10–25%, and protein content of 40–60% [62].

On the other hand, Microcystis aeruginosa, detected in the biomass collected in August, is a widespread cyanobacterium capable of forming water blooms that can lead to the production of microcystins—cyclic peptides with hepatotoxic properties. Microcystins are resistant to degradation and can bioaccumulate in aquatic organisms, posing a risk to human and animal health. Their presence in organic substrates intended for methane fermentation can pose a health risk to biogas plant operators and affect the quality of the biogas produced [63]. Previous studies have shown that methane fermentation processes can lead to the biodegradation of microcystins, but the effectiveness of this process depends on many factors, such as pH, temperature, microflora composition and residence time. Preliminary results indicate that suitable process conditions may boost the degradation of these toxins [64]. In the context of biosafety, it is important to monitor the presence of Microcystis aeruginosa in feedstock supplied to biogas plants and to assess the potential risks associated with microcystin production. The studies presented have not shown any negative impact of this species on the efficiency of anaerobic digestion.

In September, with the observed shift to a more diversified taxonomic structure (diatoms—39%, dinoflagellates—39%, and cyanobacteria—32%), the yield of CH₄ decreased to 229 ± 9 mL/g VS, and biogas production to 386 ± 12 mL/g VS (Table 3 and Table 4, Figure 2). The CH₄ content of the gas reached 59.3 ± 1.2%, and its maximal production rate reached 25.3 ± 1.3 mL/g VS·d, with k = 0.11± 0.01 1/d (Table 4). Although the contents of saccharides (530 ± 36 mg/g TS) and lipids (95 ± 10 mg/g TS) remained high, the increased contribution of diatoms with their siliceous frustiles could diminish substrate availability in the initial stage of hydrolysis (Table 3 and Table 4). In addition, increased contents of TN (6.3 ± 0.4% TS) and protein (375 ± 32 mg/g TS) at the reduced TOC content and C/N indicate the substrate to be more balanced but less chemically reactive (Table 3).

In October, when diatoms prevailed in the phytoplankton structure (76% TS), the efficiency of anaerobic digestion decreased noticeably (Table 3), leading to the lowest biogas yield across the season (355 ± 13 mL/g VS) and to CH₄ production of 201 ± 8 mL/g VS, at CH₄ content of the biogas reaching 56.8 ± 1.7% (Table 4, Figure 2). The methane production rate was the lowest (18.0 ± 1.0 mL/g VS·d), despite relatively high contents of protein (438 ± 44 mg/g TS) and TN (7.1 ± 0.6% TS), (Table 3 and Table 4). These decreases could be due to the presence of mineral fractions (siliceous frustules), low contents of lipids (83 ± 9 mg/g TS) and carbohydrates (477 ± 32 mg/g TS), as well as lower contents of TOC (44.5 ± 1.6% TS) and VS (75.0 ± 1.6% TS), (Table 3). The kinetic constant (k = 0.11 ± 0.01 1/d) indicates an extended preincubation period and diminished substrate bioavailability.

To recapitulate, the production yields of biogas and CH₄ are strongly correlated with the taxonomic composition of the microalgal biomass. An overview of literature indicates the highest recorded biogas production of 740 mL/g VS from Spirulina platensis [65] and 615 ± 7 mL/g VS from Laminaria sp. [14]. In turn, the average CH₄ yield from microalgae was reported to range from 227 mL/g VS from green algae to 262 mL/g VS from brown algae [66]. The biogas and methane yields obtained in the present study fit within the range of reference values. Examples of the efficiency of anaerobic digestion based on the use of microalgal biomass as an organic substrate and the basic parameters of this process are shown in

Table 6. Examples of efficiency and basic parameters of anaerobic digestion based on the use of microalgal biomass as an organic substrate.

Taxon	Biogas/CH4 Content	Methane Content (%)	T (°C)	OLR (g VS/L·d)	HRT (Days)	Reference
Durvillea antarctica	179.3 ± 80.2 mL CH4/g TS·d	–	37	3	1	[67]
Macrocystis pyrifera + Durvillea antarctica	164.2 ± 54.9 mL CH4/g TS·d	–	37	3	1
Macrocystis pyrifera	181.4 ± 52.3 mL CH4/g TS·d	–	37	3	1
Chlorella vulgaris	240 mL CH4/g VS	–	35	1.0	28	[68]
	150 mL CH4/g VS	–	35	1.0	16
Spirulina maxima	240 mL CH4/g VS	–	35	1.0	33	[69]
Arthrospira platensis	481 ± 13.8 mL CH4/g VS	61	38	–	–	[70]
Euglena gracilis	485 ± 3 mL biogas/g VS	67	38	–	–
Chlorella kessleri	335 ± 7.8 mL biogas/g VS	65	38	–	–
Dunaliella salina	505 ± 24.8 mL biogas/g VS	64	38	–	–
Chlamydomonas reinhardtii	587 ± 8.8 mL biogas/g VS	66	38	–	–
Phaeodactylum tricornutum	800 ± 30 mL/L·d	78.6 ± 5.0	33 ± 2	1.9	1.9	[71]
	800 ± 30 mL/L·d	75.1 ± 8.9	33 ± 2	1.9	1.9
Scenedesmus obliquus	287 ± 10.1 mL biogas/g VS	62	38	–	–	[70]
	600 ± 20 mL biogas/L·d	77.1 ± 3.9	33 ± 2	2.8	2.2	[71]
	400 ± 0 mL biogas/L·d	74.3 ± 2.5	33 ± 2	2.8	2.2
Scenedesmus sp. + Chlorella sp.	818 ± 96 mL biogas/L·d	–	35	6.0	10	[72]
	573 ± 28 cm3 mL biogas/L·d	–	35	4.0	10
	180 ± 8 mL biogas/L·d	–	35	2.0	10

.

A crucial element that usually determines the potential implementation of new technological solutions is their ultimate energy efficiency and cost-effectiveness. These practical aspects should also be considered when evaluating the use of phytoplankton from natural, eutrophic waters such as the Gulf of Gdansk. Based on previous research by the authors, conducted on a semi-industrial scale and evaluating the energy efficiency of a system for harvesting microalgal biomass from the Vistula Lagoon, it can be concluded that such a solution can be economically viable. It has been shown that a net electricity of 4.69 kWh and a potential net energy of 23.23 kWh can be achieved by using a suitable method for separating the biomass and the technological parameters of the anaerobic digestion process. In the least favorable variants, the energy balance was negative, reaching −0.79 kWh of electricity [12]. However, it should be noted that when using phytoplankton biomass from natural waters, both a positive energy balance and the ecological benefits—such as the removal of organic matter from the ecosystem, mitigation of algal blooms, reduction in eutrophication, and potential restoration of degraded water bodies for economic and recreational purposes—should be considered. This necessitates a comprehensive Life Cycle Assessment (LCA).

3.5. Dependencies and Correlations

To assess the influence of selected chemical parameters of phytoplankton biomass from the Gdansk Bay on methane fermentation efficiency, a linear correlation analysis was conducted between biogas and methane yields and specific chemical indicators of the biomass composition (Figure 3). High coefficients of determination (R²) indicate strong dependencies between the composition of the analyzed organic substrate and the efficiency of the biomethanation process.

The strongest correlations were found between the total organic carbon (TOC) content of the biomass and both biogas yield (R² = 0.9435) and methane yield (R² = 0.9651), (Figure 3a). The corresponding linear regression equations indicate a significant positive effect of increasing TOC content on anaerobic digestion efficiency (Figure 3a). These results are consistent with the literature findings, where TOC was considered a general indicator of the bioavailable organic matter for microbial conversion [73]. Higher TOC values may reflect the presence of readily biodegradable fractions, including lipids and carbohydrates, which promote rapid degradation and CH₄ production [74].

High R² values determined for the C/N ratio, specifically for biogas production (0.9192) and methane production (0.9326) (Figure 3b), confirm the significant role of this parameter in controlling the stability of methane fermentation [75]. Previous studies have proved that a too low C/N ratio can lead to surplus ammonia production, which is toxic to methanogenic microorganisms, and that a too high C/N ratio reduces nitrogen availability necessary for enzyme synthesis [76]. An optimal C/N range aids balanced growth of fermentative and methanogenic bacterial populations, further reflected in higher methane yields [77]. In the present study, the C/N ratio ranged from 6.3 ± 0.3 to 11.4 ± 0.6 (Table 3), which is consistent with a typical C/N ratio for microalgae, i.e., 4–10 [78]. However, these values are lower than those recommended for anaerobic digestion processes (25–30) [79]. The regression equations obtained in this study suggest that each unit increase in the C/N ratio corresponded to an approximate increase in CH₄ production of 13 mL/g VS, which may have practical implications for optimizing co-fermentation substrate mixtures [80].

Protein content showed a strong negative correlation with biogas production (R² = 0.8999) (Figure 3d), supporting the hypothesis that high concentrations of nitrogenous compounds can lead to acidification of the environment and increased ammonium ion levels [80]. Although the correlation with CH₄ yield was also negative (R² = 0.8700), its strength was somewhat lower (Figure 3d). The regression equation indicates that an increase in protein content by 100 mg/g TS could result in a reduction in biogas production by up to 43.6 mL/g VS. This observation is consistent with findings from previous studies showing the limited biodegradability of protein fractions and their potential inhibitory effect [81]. In the case of the lipid content, a strong positive correlation was observed with biogas yield (R² = 0.9277), reflecting the high energy value of this fraction, more than twice that of carbohydrates and proteins (Figure 3e). The regression equation suggests that an increase in lipid content by 100 mg/g TS results in an increase of approximately 168 mL/g VS in biogas production. Although the correlation with CH₄ was slightly weaker (R² = 0.8700), it still confirmed the positive impact of lipids (Figure 3e). It should be noted, however, that excessive lipid content can lead to the accumulation of long-chain fatty acids (LCFAs), which are more difficult to degrade and may disrupt the methanogenic microbiota [82].

The lowest R² values were determined for carbohydrate concentrations, both compared to biogas (R² = 0.8118) and CH₄ (R² = 0.8700) (Figure 3f). Although carbohydrates are readily available energy sources for fermentative bacteria, their rapid fermentation can cause sudden acidification and the production of volatile fatty acids, disturbing process stability [83]. The lower correlation strength observed may result from high variability in sugar fraction availability across the analyzed phytoplankton biomass samples.

Seasonal variability and the associated plankton succession in temperate estuarine ecosystems significantly influence the chemical composition of algal biomass, leading to differences in biogas production [21]. Different genera of algae can store various amounts of carbohydrates, proteins, and lipids, potentially affecting biogas yields. Ample studies have indicated a correlation between methane production efficiency from eukaryotic microalgae and their biochemical composition, including particularly their macronutrient content [84]. However, recent studies suggest that macronutrient ratios alone are not the key determinants of methane yield, and that algal biomass composition should not be the sole criterion for selecting the best microalgal strains for anaerobic digestion. More critical factors include algal biomass production rates, cell wall structure, and the potential for co-fermentation with other organic substrates [60].

4. Conclusions

Significant changes in the taxonomic structure of the phytoplankton were observed during the growing season in the waters of the Gulf of Gdansk, ranging from the dominance of green algae and diatoms in the spring (May–June) to intensive cyanobacterial blooms in the summer months (July–August), and the predominance of diatoms in autumn (September–October). In addition to this taxonomic succession, notable fluctuations in the chemical composition of the phytoplankton biomass were also observed. The cyanobacterial biomass exhibited the highest content of organic compounds, while the lowest total nitrogen (TN) values were obtained. Thus, the chemical composition of the biomass proved to be a key factor influencing the efficiency of methane fermentation.

Seasonal variations in the structure and composition of the biomass directly influenced anaerobic digestion performance. The highest biogas and methane yields were observed in August, coinciding with the highest methane production rate and the highest methane content in the biogas. The correlation analysis indicated that total organic carbon (TOC) and C/N ratio are the most important indicators of methane fermentation efficiency of phytoplankton biomass. These findings suggest that seasonally harvested phytoplankton biomass can serve as a valuable and promising substrate for biomethane production.