Impact of Thermo-Mechanical Pretreatment of Sargassum muticum on Anaerobic Co-Digestion with Wheat Straw

Abstract

Sargassum muticum (SM) is an invasive macroalgal species seasonally occurring in large quantities. While generally suitable for anaerobic digestion, recent studies resulted in low specific methane yields (SMYs), presumably due to salt, polyphenol, and high fiber contents of this marine biomass. In this study, the specific biogas yield (SBY) and SMY of SM alone as well as in co-digestion with wheat straw (WS) were investigated in batch tests at process temperatures of 44 ± 1.4 °C with a retention time of approx. 40 d. The pretreatment variants of SM were examined with regard to desalination and disintegration to potentially improve digestibility and to enhance the overall performance in anaerobic digestion. A sole mechanical treatment (pressing) and a thermo-mechanical treatment (heating and pressing) were tested. Batch assays showed that pressing increased the SMY by 15.1% whereas heating and pressing decreased the SMY by 15.7% compared to the untreated variant (87.64 ± 8.72 mL/g_VS). Both anaerobic digestion experiments generally showed that co-digestion with WS can be recommended for SM, but the observed SBY and SMY were still similar to those of other studies in which SM was not pretreated. The mechanical pretreatment of SM, however, offers the potential to enhance the SMY in the anaerobic digestion of SM with WS, but further research is necessary to identify the optimum upgrading approaches since the overall SMY of SM is relatively low compared to other substrates that are commonly used in anaerobic digestion. In addition to anaerobic digestion, SM as an already available biomass could also be of interest for further utilization approaches such as fiber production.

1. Introduction

In the last few decades, apparently limitless access to cheap fossil energy sources has enabled economic growth and prosperity. In the future, high energy demand must also be met but at the same time, dependencies on fossil resources as well as on certain states are problematic. Geopolitical tensions and war demonstrate how politics and energy supply are linked (e.g., in the EU). Therefore, alternative resources for energy provision and material use are of more and more interest [1]. However, not only are political aspects requiring a fundamental rethinking but to a large extent also the advancing climate change and natural limitations of fossil energy sources [2]. Transformation toward a renewable circular economy is one of the major challenges that humanity is facing. Therefore, alternative sources to generate heat, electricity, and fuels while focusing on a maximum level of sustainability are crucial. Renewable and biomass-based energy production via anaerobic digestion can generate all of the three above-mentioned forms of energy [3]. Currently, however, energy crops in combination with slurry are mainly used in anaerobic digestion, especially in Germany but also worldwide. Since the use of energy crops is criticized not only due to competition for land (food production) but also because of more frequent crop failures connected with climate change [4,5], alternatives must be explored here as well. In addition, the oceans are experiencing an ever-increasing nutrient input from various sources such as agriculture. As a result, invasive algae species (including Sargassum spp.) are spreading more rapidly which is harmful to native organisms and thus represents a threat to the ecosystems of whole geographical regions [6,7]. Despite criticism of the use of terrestrial biomass, marine biomass—especially macroalgae—still remains an underutilized biomass with great potential for energy production [8]. The marine environment contains approx. 80% of living biomass and contributes about 50% to net global biomass production [4]. Thus, the anaerobic digestion of invasive algae could help to close nutrient cycles, reduce land use pressure, improve environmental conditions (of the sea), and at the same time provide renewable energy. In the first step, the algae would extract nutrients from the eutrophic waters, secondly, energy could be produced in anaerobic digestion from the harvested algae, and finally, nutrients contained in the digestate can be returned to the agricultural cycle [9]. McHugh [10] also mentions that the use of algae fertilizer is beneficial because it increases seed germination and improves frost resistance as well as resilience to fungal and insect attacks.

Many different algae species exist that can be classified into three categories based on their appearance, namely, green, red, and brown algae. In terms of the biomethane potential (BMP) described by Murphy et al. [11], Laminaria spp. (brown algae) and Ulva spp. (green algae) are the most studied species in the past few years [8]. However, according to Kusek et al. [7], the brown algae Sargassum spp. is one of the most abundant macroalgae genera in the world’s oceans, with more than 300 subspecies [12]. In 2018, about 20 million tons of this algae harmed the waters as well as the coasts of the Tropical Atlantic Ocean, the Caribbean Sea, the Gulf of Mexico, and the east coast of Florida [7]. In addition, it is also found in Europe along the coasts from Norway to Portugal, and unlike native species, it spreads invasively [13]. Originally, the subspecies S. muticum—which is the main focus of this study—originates from the northwest Pacific of Japan, where it is used for the purposes of aquaculture or alginate production [14]. Few studies have been examining different macro-algae species in terms of their BMP. Nevertheless, for S. muticum, a dry matter (DM) content of 171 g/kg based on fresh matter (FM) and a volatile solid (VS) content of 634 g/kg_DM was found [8]. Furthermore, a lower specific methane yield (SMY) compared to other algae species was measured in previous studies (e.g., 0.13 L/g_VS for S. muticum versus 0.24 L/g_VS for brown algae Undaria pinnatifida [8]) whereas Maneein et al. [15] found an SMY ranging between 0.13 L/g_VS and 0.17 L/g_VS for freeze-dried and extensively washed S. muticum. The relatively low SMY could be explained by inhibitors or it could be caused by a lower overall digestibility of the algal genus. Jard et al. [8], for instance, found a fiber content of 512 g/kg_DM in S. muticum, which was the highest fiber content compared to nine other algae analyzed in this study. They measured the total fiber content via the enzymatic–gravimetric method. The exact nature of the fibers was, however, not specified, but they indicated a more difficult degradability due to the overall higher fiber content. Furthermore, they also found relatively low levels of sugars (166 g/kg_DM) and proteins (84 g/kg_DM) for S. muticum compared to other algae. Moreover, they also found high levels of polyphenols (mainly phlorotannin [16]), which can inhibit the growth of microorganisms [17] and destroy their cell walls [18]. In addition, sea algae can lead to increased salinity in digesters, which could negatively impact the different stages of the anaerobic digestion process [11].

The SMY of substrates is mainly influenced by their proportions of proteins, fats, and carbohydrates. The composition of Sargassum spp. can vary drastically depending on parameters such as harvesting location or season [9]. Marquez et al. [19] showed that different Sargassum spp. consist of 0.82–8.2% of lipids, 0.86–22.38% of proteins, and 4.83–17.9% of fibers (depending on the reference unit) whereby some of those values are based on samples with residual moisture content. Moreover, Monlau et al. [20] reported concentrations of 66%_DM for carbohydrates in S. fulvellum whereas Borines et al. [21] showed fiber compositions for 70 °C dried samples of Sargassum spp. consisting of 20.35% alphacellulose, 25.73% hemicellulose, 46.08% holocellulose (cellulose and hemicellulose), and 5.04% mannitol. Thus, the hemicellulose content is comparable to that of wheat straw, which is in a range of 20–33.8%_DM [22]. Borines et al. [21] did not specify the exact structural composition of these fibers, but stated that for bioethanol production from Sargassum spp., pretreatment is required due to a higher amount of hemicellulose than alphacellulose. The aspect of different levels of biodegradability for different S. muticum components could also be an important aspect for anaerobic digestion.

According to the literature, the C:N ratio for a proper microbial environment should be around 30:1 [3]. Since Soto et al. [9] found a C:N ratio close to 10:1 for three different S. muticum samples, co-digestion with other substrates such as wheat straw could be beneficial as wheat straw has a higher C:N ratio of approx. 81:1 [23]. In addition, co-digestion with wheat straw would reduce the total salinity. Two previous studies have also shown that the anaerobic digestion of the brown algae Saccharina latissima with wheat [24] or wheat straw [3] had a positive effect on the resulting SMY. Furthermore, the study of [25] also shows that co-digestion generally has a positive effect on the SMY during the anaerobic digestion of S. muticum.

Based on the arguments above, it is important to improve the utilization efficiency of S. muticum, especially with regard to its performance in anaerobic digestion (e.g., in terms of the SMY or optimum substrate mixtures). Via this, S. muticum could become an economically attractive feedstock and a resource for energy and material use while simultaneously reducing damage to ecosystems. Thus, the objectives of the presented study were to investigate and improve the SMY of S. muticum in co-digestion with wheat straw by applying different pretreatment methods compared to an untreated variant. In addition, the SMY of each substrate without pretreatment was measured.

To the best of the authors’ current knowledge, previous BMP tests involving Sargassum spp. have been conducted as batch tests in the mesophilic temperature range at around 37 °C. Since higher temperatures might lead to a better degradability of the substrate, this study aimed to investigate an intermediate temperature range between mesophilic and thermophilic conditions of 44 °C. A mixture of two inoculum types (mesophilic and thermophilic) was therefore used for all variants.

2. Materials and Methods

2.1. Substrates, Inocula, and Experimental Approach

S. muticum was collected at several locations in the Sylt–Rømø Bay, German–Danish North Sea coast (55°01′51.3″ N 8°26′01.3″ E) (Figure 1), during mid-June 2022 by employees of Alfred Wegener Institute (Bremerhaven, Germany). After collecting several kg of the fresh algae, they were rinsed with fresh water to remove adhering sand and salt. Before delivery to the University of Applied Forest Sciences Rottenburg (HFR, Rottenburg am Neckar, Germany), the algae were dried for 16–18 h at a drying temperature of 60 °C for safe storage. Before further treatment, the algae were manually chopped into pieces of 2–4 cm in length with a customary knife. In the further course of this study, S. muticum is referred to as SM.

Wheat straw was used as a co-digestion substrate and it was collected at an agricultural farm in Bad Buchau, Germany. After collection, the wheat straw was packed in plastic bags and transported to HFR. Since lignocellulosic material is difficult to degrade in anaerobic digestion, the wheat straw was crushed and partially defibered via a cutting mill (Pulverisette 19, Fritsch GmbH, Idar-Oberstein, Germany) to a particle size of 4 mm. In the further course of this study, wheat straw is referred to as WS. All digestion experiments were performed based on 60 °C dried SM and sun-dried WS as initial materials. Thus, all materials contained residual moisture but the DM concentration (absolute dry) of all substrates was additionally measured as described in the following sections.

Since anaerobic digestion experiments were carried out at 44 °C, which represented an intermediate temperature range between mesophilic and thermophilic, two types of inocula were used in a defined mixture. Firstly, thermophilic inoculum was provided by a local and full-scale biogas plant in southern Germany, which operates at a process temperature of 50 °C while being based on energy crops and cow manure, including wheat straw. The digestate was separated into a liquid and solid phase at the farm, and only the liquid phase was used as inoculum in this study. Secondly, mesophilic digestate was collected from the State Institute of Agricultural Engineering and Bioenergy (University of Hohenheim, Stuttgart, Germany), where an own and defined inoculum is constantly available. This inoculum is characterized by low residual gas potential and kept at 37 °C in a 400 L reactor [26]. The digestate was sieved (mesh size 1 mm) and the liquid phase was used as inoculum. The two inocula were mixed in equal shares for the anaerobic digestion experiments. Through this procedure, a variety of mesophilic and thermophilic microorganisms were provided for the anaerobic digestion experiments with SM and WS.

The overall procedure of the experiments can be divided into three steps that analyzed:

(1)

The impact of co-digestion of SM + WS on the specific biogas yield (SBY) during 40 d of anaerobic digestion by using the Hohenheim Biogas Yield Test (HBT) procedure. Therefore, different variants with SM, WS, and SM + WS as co-digestion were conducted.

(2)

Two different pretreatment methods to enhance the substrate characteristics of the brown algae SM in co-digestion with WS in order to achieve increased SBY and SMY by reducing salinity and by enhancing disintegration. Therefore, a batch assay with increased volumes (compared to HBT) of 2 L glass vessels was carried out.

(3)

A second HBT series with the variants mentioned in (1) but with the inocula residues (digestates) of the 2 L batch assay (2), to analyze if the microbes have adapted to the new substrate to ultimately achieve higher SBY and SMY.

Only SM was mechanically and thermo-mechanically pretreated. Thus, the mixtures of SM + WS are always based on untreated or pretreated SM and WS without pretreatment (except for crushing as stated).

2.2. Pretreatment Methods for SM

For all pretreatment variants used in a 2 L batch system as described in Section 2.3, SM was rewatered overnight at a 7.4:1 ratio to recreate the original FM condition (FM₁). After soaking or re-hydration, the algae were drained in a kitchen sieve (mesh size 4 mm). The re-hydration ratio was found to be 3.8:1, which showed that SM samples did not reach their initial FM₁, but a lower FM₂. In the following, results are related to FM₂ after re-hydration. Pretreatment procedures are further described and shown in Figure 2.

2.2.1. Mechanical Pretreatment—Pressing

Samples of 265 g of drained SM were pressed at a pressure of 2.3 bar for approx. 30 s. For this purpose, a customized pressing system was used, whereby the pressure was adjusted through a defined force and area. During this procedure, an average of 34 ± 7.4 g of liquids was removed. The remaining solid substrate, referred to as SM_pressed, was used for anaerobic digestion.

2.2.2. Thermal Pretreatment—Heating and Pressing

For thermal pretreatment, samples of 265 g of drained SM were filled in beakers and placed in a sterilizer (DX-45, Systec GmbH & Co. KG, Linden, Germany). In the beginning, a vacuum was generated using three vacuum pulses. Then, the vacuum was kept, and the samples were heated up to 121 °C. After a holding time of 20 min, the produced vapor was quickly released until atmospheric pressure was reached. The sterilizer kept the final temperature of 101 °C for 10 min until the samples were removed. After thermal treatment, the samples were pressed as described above, whereby they lost an average amount of 14 ± 3.4 g of liquid. The remaining solid substrate, referred to as SM_{heated and pressed}, was used for anaerobic digestion.

Both press waters of sole mechanical and thermo-mechanical pretreatment, as well as the soaking water from re-hydration, were collected and later analyzed for their conductivity as well as for their elemental profiles through inductively coupled plasma-optical mass spectrometry (ICP-OES) analysis.

2.3. Digestion Systems

For anaerobic digestion experiments in this study, two different digestion systems were used: firstly, HBT (according to Hülsemann et al. [26]) and secondly, an anaerobic digestion system using 2 L glass vessels (Figure 3). Those two anaerobic digestion systems were selected because the smaller format of the HBTs allowed for the additional investigation of the SBY of the individual substrates during anaerobic digestion. In the 2 L variants, only mixtures of SM + WS were used. Furthermore, the number of replicates were increased through the HBT experiments, and the results of both digestion systems were validated by each other. For the 2 L digestion experiment, the term “2L-DE” will be used in the further course of this study.

2.3.1. 2L-DE

Various hose connections and an agitator are integrated into the bottle cap of the anaerobic digestion reactors (Figure 3). One hose is connected to a glass column that contains a saturated saltwater solution, whereby the absolute biogas yield is determined by the water level. The produced biogas is then released into 10 L gas bags (Plastigas, Linde GmbH, Pullach, Germany). The glass column can store a total gas volume of 1 L, if more is produced, the gas can escape through an overpressure hose which is a safety mechanism that is not needed in normal operation since gas losses can be prevented through regular and frequent readings. In the course of the experiment, biogas composition was analyzed four times via a portable gas monitor (Biogas 5000, Geotech, Coventry, UK) with regard to methane concentrations.

SM is an algae species with floating bodies which can promote the formation of floating layers during anaerobic digestion; therefore, an inclined axis agitator should obtain a better homogenization of the biomass and avoid the formation of floating layers. Thus, the digesters were mounted at an angle of 45° and were automatically stirred for 2 min every 2 h. For BMP determination, a total number of 12 insulated glass vessels were kept at 44 ± 1.4 °C via a thermostat-controlled heating mat.

The batch experiment was used to investigate pretreatment methods of SM in co-digestion with WS, especially regarding SBY and SMY. All anaerobic digestion variants were tested in triplicate and also included inoculum-only variants (blanks). For each digester, the experimental set-up of the 2L-DE is presented in

Table 1. Experimental set-up of 2L-DE presenting the mixtures consisting of the inocula mixture (mesophilic, thermophilic) in equal shares, wheat straw (WS), and Sargassum muticum (SM). The table also shows the contained mass input of dry matter (DM) and volatile solids (VS) for each digester number (Digester No.). The amount of fresh matter (FM₂) of each substrate is additionally presented. FM₂ is further explained in Section 2.2.

Substrate	FM2	DM	VS	Digester No.
	g	g	g	(-)
Inoculum	-	84.8 ± 1.9	54.7 ± 1.9	1–12
WS	-	47.1 ± 0.3	44.05 ± 0.3	4–12
SM	265 ± 0.12	61.3 ± 0.1	41.35 ± 0.1	4–6
SMpressed	226 ± 6.3	58.9 ± 6.3	39.7 ± 6.3	7–9
SMheated and pressed	230 ± 3.9	57 ± 3.9	39.3 ± 3.9	10–12

.

One week before the start of the experiment, the inoculum mixture was filled into the glass vessels (overall 1500 g in equal shares), flushed with nitrogen, and kept on starvation during this time. It was steadily heated up from an initial temperature of 37 °C to 44 °C. In this study, the absolute biogas production (mL) was determined twice a day for the first 17 d and after that, it was determined on a daily basis.

For evaluation, SBY and SMY were calculated based on standard conditions (dry gas, 1013 hPa, 0 °C) whereas the following formulas represent the calculation of the gas yields. In this study, the anaerobic digestion experiments were evaluated based on substrate-specific gas yields. SBY of SM + WS without the yield contributed by the inoculum (I) was calculated as

$${SBY}_{SM,WS}=\frac{{BG}_{DC}-{BG}_I}{m_{VS, SM}+m_{VS, WS}},$$

(1)

where SBY_SM,WS (mL/gVS_{SM, WS}) is the specific biogas yield from SM and WS, and BG_I (mL) is the specific biogas yield from inoculum alone.

The specific methane yield (SMY) of the inoculum blanks was calculated as

$${SMY}_I={{\sigma }_I·SBY}_I ,$$

(2)

where SMY_I (mL/gVS_I) is the specific methane yield from the blanks, SBY_I (mL/gVS_I) is the specific biogas yield of the inocula, and σ_I (-) is the analyzed volumetric methane concentration in the biogas produced by the blanks.

SMY from the SM and WS mixture was calculated as

$${SMY}_{SM,WS}=\frac{{ {\sigma }_{DC}·BG}_{DC }-{{\sigma }_I·BG}_I}{m_{VS,SM}+m_{VS,WS}}$$

(3)

where SMY_SM,WS (mL/gVS_SM,WS) is the specific methane yield from SM and WS. Since methane concentrations were measured four times during the experiment based on the entire digester content, the determined individual concentration was used for the respective period of time. A weighted average was finally calculated for each variant.

2.3.2. Hohenheim Biogas Yield Test (HBT)

Both HBT series 1 and 2 were executed according to VDI 4630 and Hülsemann et al. [26]. They consisted of 12 × 100 mL glass syringes that included a scale to manually record the biogas volume (SMY was not determined in this experiment). The syringes were stored for 40 d in a heating cabinet at 44 °C and they were moved manually to homogenize the substrate (HBT 1: twice a day for 30 s per syringe and HBT 2: once a day for 30 s). The experimental set-up is shown in

Table 2. Experimental set-up of Hohenheim Biogas Yield Test (HBT) for each syringe (S) presenting the mixtures containing inoculum, sun-dried wheat straw (WS), and Sargassum muticum (SM) on storage-dry basis as well as VS ratio of substrate to inoculum.

Variants	Unit	Blanks	SM	WS	SM + WS
		S 1–3	S 4–6	S 7–9	S 10–12
SM	g	-	0.5 ± 0.01	-	0.3 ± 0.01
WS	g	-	-	0.51	0.2 ± 0.01
Inoculum	mL	30	30	30	30

. The mixing ratio of SM and WS was approx. the same as in the 2L-DE.

For HBT 2, the initial inocula mixture was not used but the liquid phase of the inocula contained in digester 8 of the mechanical pretreated algae variant from the 2L-DE was selected. This procedure was chosen because full-scale biogas plants usually operate based on a continuous feeding process (e.g., typical for stirred tank reactors). Therefore, the HBT 2 experiment was selected to investigate whether salt inhibition can be observed when the inoculum, which has an already higher salt content due to the previous fermentation of SM, is used again. Furthermore, the liquid phase of digester 8 was chosen as this digester had produced the highest SMY in 2L-DE. It should be noted that this inocula probably had a higher residual gas potential compared to typical inocula as used according to VDI 4630.

2.4. Laboratory and Statistical Analyses

2.4.1. Dry Matter Determination

DM determination was performed on storage-dry SM and WS substrate via a six-fold determination according to DIN EN ISO 18134-1. Therefore, 6 × 1 g of each grounded substrate (1 mm) was dried for 24 h at 105 °C (Drying oven UN450, Memmert GmbH & Co. KG, Schwabach, Germany).

2.4.2. Ash Content

Ash content determination was carried out using the standard DIN EN 14775. The procedure was based on DM. An amount of 1 g of each sample was weighed into four ceramic vessels and incinerated in a muffle furnace (AAF1100, Carbolite Gero GmbH & Co. KG, Neuhausen, Germany). The furnace was operated with a predefined program that raised the temperature to 250 °C within 30–50 min, with a holding time of 1 h. The temperature was then further increased to 550 °C within 30 min and held for at least 2 h. Until the samples were reweighed, they remained at 105 °C in the oven.

VS contents of each sample were then calculated based on the mean value of the determined ash concentration (VS = 100% − mean_ash).

2.4.3. Elementary Analysis (CHNO) and Theoretical (Stoichiometric) Biogas Potential

Contents of C, N, and H were determined in a six-fold detection with an elementary analyzer (CHN828, LECO Corporation, St. Joseph, MI, USA). Each sample (approx. 0.1 g_DM per repetition) was weighed into foil cups and formed into a tear shape. In the analyzer, samples were incinerated at 1050 °C and the resulting gases were measured for their C, H, N content. In this method, the O value is calculated by the difference to 100% where the ash content is included.

Theoretical biogas and methane yield of SM and WS were determined with Formula (4) of Buswell and Müller which was modified by Boyle [27] and expressed at standard conditions (1013 hPa, 0 °C, dry gas).

$${ C}_n{H}_a{O}_b{N}_c{S}_d + (n-\frac{a}{4}-\frac{b}{2}-\frac{3}{4} c-\frac{d}{2}){ H}_{2}O \to (\frac{n}{2}-\frac{a}{8}- \frac{b}{4}-\frac{3}{8} c-\frac{d}{4}) C{O}_2+(\frac{n}{2}-\frac{a}{8}- \frac{b}{4}-\frac{3}{8} c-\frac{d}{4}) C{H}_4+cN{H}_3+d{H}_{2}S$$

(4)

2.4.4. Multi-Elemental Determination

An amount of 400 ± 100 mg_DM of each milled biomass samples (cutting mill pulverisette 19, Fritsch GmbH, Idar-Oberstein, Germany, sieve 1.0 mm) was placed in a digestion vessel, mixed with 1.0 mL H₂O₂, and shaken for 3 s. After 5 min, 2.0 mL of HNO₃ was added and shaken again for 3 s. After 5 min, another 2.0 mL portion of HNO₃ (65%) was added. After 30 min, 3.0 mL HCl (35%) was added, and the digestion vessels were closed and left overnight for 14 h [28]. The next day, 6.0 mL HCl was added, and the reaction mixture was digested in a microwave oven (Microwave GO, Anton Paar Ltd., Saint Albans, UK). The microwave digestion program heated up to 175 °C within 20 min and a holding time of 30 min. Then, it was heated up again to 185 °C within 5 min and held for another 5 min. After cooling the digestion vessels, the samples were filled up to 50 mL with double-distilled water. These different solutions were measured in the ICP-OES system (Spectroblue TI with a Cetac Ltd. Autosampler ASX-260, Ametek Ltd. Spectro, Leicester, UK and Teledyne CETAC Technologies, Omaha, NE, USA). ICP-OES analyses were carried out in a six-fold determination and expressed based on mean values ± standard deviation (g/kg_DM for WS, SM, and inoculum; mg/L for press waters).

2.4.5. Gross Calorific Value (GCV) and Sulphur Chloride Determination

The GCV was determined according to DIN EN 14918 with an isoperibol bomb calorimeter (calorimeter model: C6000, bomb model: C601012, IKA GmbH & Co. KG, Staufen, Germany) by using approx. 1 g_DM of SM and WS. A mean value including standard deviation was then calculated based on four replicates per sample.

At the end, the residues in the bomb were washed off with double-distilled water and collected in analytical tubes. Then, the tubes were filled up to 50 mL with double-distilled water and Cl values were analyzed with an ion chromatograph (883 basic IC Plus, Metrohm AG, Herisau, Switzerland).

2.4.6. Statistical Analysis

The results of the SBY and SMY per variant were analyzed via one-way analysis of variance (ANOVA) with α = 0.05. It was performed to compare the effect of pretreatment methods (dependent variable) on SBY and SMY (independent variable). The experiments were carried out in triplicate, which is why the obtained results are presented with mean values, including their determined standard deviation.

3. Results and Discussion

3.1. Characteristics of Samples Used

The biochemical composition of the initial substrates (SM, WS, and inocula), the residual moisture content (based on storage-dry substrate of SM and WS) as well as the inoculum composition of D8 (after the 2L-DE) are given in

Table 3. Biochemical composition of the samples used, ratio of volatile solids (VS), ash content, elementary analysis, and gross calorific value (GCV) based on dry matter (DM) of Sargassum muticum (SM) and wheat straw (WS) as well as residual moisture content of storage (60 °C)-dry substrate.

Parameter	Unit	SM (Untreated)	WS	Inoculum Thermophilic	Inoculum Mesophilic 1	Inoculum Mixture D8
DM	%FM	11.82 ± 0.47	-	6.73 ± 0.45	4.4 ± 0.0	NA
VS	%DM	67.45 ± 0.42	93.59 ± 0.33	65.89 ± 0.47	61.7 ± 0.1	NA
Residual moisture	%DM	12.34 ± 0.47	11.19 ± 0.59	-	-	-
Ash	%DM	32.55 ± 0.41	6.41 ± 0.08	NA	1.7 ± 0.0 (%FM)	NA
C	%DM	31.94 ± 0.27	46.95 ± 0.16	NA	NA	NA
H	%DM	5.17 ± 0.11	6.52 ± 0.03	NA	NA	NA
N	%DM	2.34 ± 0.07	0.66 ± 0.05	NA	NA	NA
GCV	kJ/gDM	12.53 ± 12	18.79 ± 18.9	NA	NA	NA
Ca	g/kgDM	10.39 ± 0.5	2.97 ± 0.17	54.90 ± 0.54	NM	28.33 ± 1.67
K	g/kgDM	75.78 ± 1.94	15.59 ± 0.24	0.02 ± 0.87	NM	116.28 ± 5.25
P	g/kgDM	1.26 ± 0.04	0.92 ± 0.03	1.79 ± 3.98	NM	5.54 ± 0.26
S	g/kgDM	7.91 ± 0.12	0.67 ± 0.02	0.99 ± 2.2	NM	10.00 ± 0.6 *
Co	g/kgDM	0.001 ± 0.00	<0.001	0.001 ± 0.03	0.003	0.002 ± 0.00
Fe	g/kgDM	2.17 ± 0.2	0.15 ± 0.02	8.98 ± 0.17	3.24	6.00 ± 0.37
Mn	g/kgDM	0.25 ± 0.01	0.04 ± 0.00	0.40 ± 4.8	0.316	0.37 ± 0.02
Mg	g/kgDM	12.95 * ± 0.31	0.68 ± 0.03	7.36 ± 0.29	NM	7.47 ± 0.36
Mo	g/kgDM	<0.001	<0.001	0.003 ± 0.09	0.007	0.004 ± 0.00
Na	g/kgDM	25.65 ± 0.68	0.14 ± 0.03	1.89 ± 0.09	NM	12.48 ± 0.65
Ni	g/kgDM	<0.001	<0.001	0.008 ± 0.14	0.015	11.04 ± 0.00
Se	g/kgDM	0.003 ± 0.00	<0.001	<0.001	0.001	<0.001
Cu	g/kgDM	0.005 ± 0.00	<0.001	0.06 ± 1.84	0.091	0.065 ± 0.00
Zn	g/kgDM	0.023 ± 0.00	0.011 ± 0.00	0.26 ± 5.71	0.378	0.400 ± 0.02
Cl	g/kgDM	104.7 ± 12.3	4.04 ± 0.18	NA	NM	NA

* calculated value outside calibration limit; NM = not mentioned; NA = not analyzed; ¹ Hülsemann et al. [26].

. The results of the elementary analysis for SM and WS, ash contents, and GCV (based on absolute-dry conditions of the samples) are presented in Table 3. The data from the mesophilic inoculum were obtained from Hülsemann et al. [26]. The drying ratio of the original 60 °C dried SM sample was 7.4:1, which corresponds to a solids content of 13.5% on a wet basis. Via the further processing of SM, a DM content of 11.82% was ultimately determined while the VS content was 67.45%_DM.

The characteristics of SM were similar to the results of previous studies dealing with S. muticum. Jard et al. [8] found a DM concentration for S. muticum of 171 g/kg_FM (17.1%_FM) and 634 g/kg_DM (63.4%_DM) of VS. Furthermore, they observed concentrations of 341 g/kg_DM (34.1%_DM) for C, 30 g/kg_DM for S, 122 g/kg_DM for Cl, and 1 g/kg_DM for P. These values are similar to those in Table 3. Borines et al. [21] did not mention which Sargassum subspecies was analyzed but they presented a residual moisture content of 11.16 ± 0.01% for 70 °C dried Sargassum spp. as well as an ash content of 26.19 ± 0.07% based on the 70 °C dried substrate. Both results are almost identical to the results of this study. Moreover, they found C concentrations of 56.81% and stated that Na, Mg, S, Zr, Ca, K, and Cl had concentrations ≤ 1% by weight (also based on 70 °C dried samples).

All values determined in this study are based on the originally used test substrate except the stated values of DM and VS for the pretreated SM. Since no retention samples of the pretreated algae were available at the start of the 2L-DE, the VS content of these variants had to be determined retrospectively. The VS and DM values presented for mechanical and thermo-mechanical pretreatment are based on SM that was re-hydrated, dried again at 60 °C, and later soaked and pretreated again. This procedure mimicked the original sampling and processing. The press waters collected during this process were analyzed with regard to VS. The relative organic losses determined were then compared to the initial SM to conclude an accurate VS content of the pretreated algae in the digestion experiments. It can, however, be assumed that fewer amounts of DM and VS are washed out with each repetition of the procedure. Therefore, it is possible that the samples that were pretreated for the digestion experiment might actually be characterized by a greater amount of washed-out DM and VS than those samples that were analyzed retrospectively. Nevertheless, it can be seen that SM contains slightly less VS when treated with a harsher pretreatment method (Table 1).

To work properly and to produce as much methane as possible during anaerobic digestion, microorganisms need a balanced supply of nutrients. Next to C, N is the most-needed nutrient, with an optimal C:N ratio in the range of 30:1 [29]. By considering only the untreated SM + WS blend (without inoculum), this resulted in C:N ratios of 38:1 in each digester. The C:N ratio of the untreated SM was approx. 14:1 whereas that of the WS was approx. 71:1. N is needed for metabolism and a stable bacterial growth, but if excessive amounts of N are present in the substrate, the biogas process can be inhibited by the increased ammonia production. Both under- and over-supply should therefore be avoided [30].

The mixing ratio of SM:WS (based on mass) was approx. 70:50. The VS ratio of the substrate (SM + WS) to inoculum was 1.56 for the untreated variant, 1.53 for the pretreatment method with SM_pressed, and 1.53 for the variant with SM_{heated and pressed}. If only the VS ratio of the SM to inoculum is considered, the following values were obtained: 0.76 for SM, 0.73 for SM_pressed, and 0.72 for SM_{heated and pressed}. Those ratios have to be considered when evaluating the results as they are higher than recommended by guidelines such as the VDI 4630.

Based on Equation (4), a theoretical and maximum possible SBY of 1093 mL/g_VS and SMY of 509 mL/g_VS for SM, respectively, and SBY of 957 mL/g_VS and SMY of 511 mL/g_VS for WS were calculated. Stoichiometric SBY and SMY are, however, not achievable in practice. In the following, the actually achieved SBY and SMY values of the experiments are shown.

3.2. Biogas Production

3.2.1. 2L-DE Results

In this study, a hydraulic retention time (HRT) of 40 d was selected instead of the termination criterion as suggested within the VDI 4630, since biogas plants often operate with a HRT of 40 d [31]. The exact HRT was 40.76 d (with regard to the last measurement).

Figure 4 shows the SMY of all variants as analyzed within the 2L-DE. The obtained mean SMY of the inoculum (35.5 ± 0.45 mL/g_VS) has been subtracted from the variants. Thus, the results shown in Figure 4 are presented without the gas production of the inoculum.

The SMY of the untreated SM + WS was 87.64 ± 8.72 mL/g_VS. It can be seen that the hydraulic pressing of SM improved the SMY in co-digestion with the untreated WS by 15.1% in comparison to the untreated SM + WS variant (Figure 5). On the other hand, an SMY reduction of 15.7% (74.75 ± 0.94 mL/g_VS) was observed for the SM_{heated and pressed} in co-digestion with WS. The untreated sample showed the highest methane concentration after the final determination of the biogas composition whereas the mechanically pretreated sample showed the lowest volumetric concentration (

Table 4. Specific biogas yield (SBY) as well as specific methane yield (SMY) of Sargassum muticum (SM) and wheat straw (WS) based on volatile solids (VS) and methane concentration.

Substrate	SBY a	SMY b	Methane Concentration in % Week
	mL/gVS	mL/gVS	1	3	5	6
Inoculum	52.8 ± 3.6	35.5 ± 0.45	59.59 ± 1.4	-	69.9 ± 0.5	72.2 ± 0.6
SM + WS	201.37 ± 30.38	87.64 ± 8.72	42.8 ± 6.3	61.9 ± 3.4	61.4 ± 0.9	62.6 ± 1.5
SMpressed + WS	220.65 ± 8.87	100.86 ± 11.37	45.9 ± 5.9	59.1 ± 2.4	61.1 ± 1.8	60.9 ± 2.3
SMheated and pressed + WS	178.67 ± 8.16	74.75 ± 0.94	39.9 ± 6.4	61.9 ± 4.7	61.3 ± 2.5	61.5 ± 1.4

Data obtained after 40 d of anaerobic digestion. ^a ANOVA analysis of SBY mean values per measured time period of untreated, mechanically pretreated, and thermo-mechanically pretreated variant resulted in P = 0.0047; F = 5.53; F_critical = 3.05. ^b ANOVA analysis of SMY mean values per measured time period of untreated, mechanically pretreated, and thermo-mechanically pretreated variant resulted in P = 0.00002; F = 11.55; F_critical = 3.05.

). The results indicate that the mechanical pretreatment achieved a positive effect on the SBY and SMY whereas the thermo-mechanical pretreatment led to a lower SMY in comparison to the untreated sample.

The experimental results as presented in Figure 4 were additionally used as input parameters for the modified Gompertz (GOM) model. Those results are included in Figure 4 while the determined and exact GOM parameters are presented as supplementary material in Appendix A.

With regard to the specific biogas production rate (BPR) per hour and for the beginning of the experiment, it can be seen that the untreated and mechanically treated variants increased their BPR to the same extent (Figure 6). Afterward, the mechanically pretreated variant increased and showed a higher BPR from day 2–7. Then, the BPR fluctuated at relatively similar levels (BPR of inoculum neglected). From day 24–33, the BPR of the thermal variant was marginally higher.

The initial intention of the pretreatment methods was to reduce the salinity of the algae and also to make it easier for microbes to degrade fibers and ultimately achieve a higher SMY.

For pretreatment, storage-dry SM was soaked with the originally determined FM ratio in tap water overnight. Since only 38.6% of the supplied water was resorbed, this procedure showed that the SM did not return to the initial FM condition. However, the soaking water of the algae as well as the press waters of the pretreated SM were examined for electrical conductivity (EC) to give an estimation of the salt content in the individual variants. Furthermore, the soaking and press waters were also analyzed via ICP-OES (

Table 5. ICP-OES analysis of soaking and press water as well as electrical conductivity (EC) of the soaking and press waters. The samples were generated via the pretreatment of Sargassum muticum.

Parameter	Unit	Soaking Water (Untreated)	Press Water (SMpressed)	Press Water (SMheated and pressed)
EC	mS	39.9	41.3	42.1
Ca	mg/L	95.89 ± 1.65 *	565.15 ± 27.85 *	341.89 ± 3.72 *
K	mg/L	1880.04 ± 25.75 *	2238.87 ± 20 *	2349.44 ± 41.46 *
P	mg/L	69.58 ± 0.23 *	82.80 ± 1.28 *	99.78 ± 2.71 *
S	mg/L	234.83 ± 1.70 *	337.41 ± 3.76 *	843.75 ± 20.92 *
Co	mg/L	0.01 ± 0.00	0.03 ± 0.00	0.04 ± 0.00
Fe	mg/L	0.88 ± 0.25	40.47 ± 3.30	39.02 ± 0.99
Mn	mg/L	13.23 ± 0.07	16.39 ± 0.22	23.16 ± 0.23
Mg	mg/L	1051.28 ± 6.71 *	1050.09 ± 9.50 *	1196.42 ± 39.93 *
Mo	mg/L	<0.001	<0.001	<0.001
Na	mg/L	1599.52 ± 8.28	2011.38 ± 12.4	2139.08 ± 36.77
Ni	mg/L	0.01 ± 0.00	0.14 ± 0.01	0.06 ± 0.00
Se	mg/L	0.04 ± 0.00	0.03 ± 0.00	0.07 ± 0.00
Cu	mg/L	<0.001	0.07 ± 0.01	<0.001
Zn	mg/L	0.13 ± 0.02	0.76 ± 0.03	0.91 ± 0.01
Cl	mg/L	NA	NA	NA

* calculated value outside calibration limit; NA = not analyzed.

). It can be seen that the EC in the press water increases with more severe pretreatment methods (untreated, to mechanical, to thermo-mechanical). Nevertheless, it has to be noted that a larger amount of press water was collected in the mechanical pretreatment (101.3 g) compared to the thermo-mechanical pretreatment (42.3 g, Figure 2). If assumed that Na is an indicator for salt content and that 1000 g of press water corresponds to 1 L, it can be calculated that the mechanical pretreatment washed out twice as much salt from the SM (203.8 mg of Na) compared to the thermo-mechanical pretreatment with 90.5 mg of Na. This could be related to the fact that through thermal heating, water evaporated, resulting in a lower moisture content in the algae and thus reducing the effect of salt leaching through subsequent pressing.

In view of the important nutrients P and S, the results showed that, via the mechanical pretreatment also, approx. twice as much P was leached out (8.4 mg) compared to the thermo-mechanical variant (4.2 mg). However, S was leached out in roughly the same amount in both variants (mechanical 34.2 mg vs. thermo-mechanical 35.7 mg (Table 5)).

3.2.2. HBT Results

The HBT tests showed that the co-digestion of SM with WS has a positive effect on the SBY (

Table 6. Specific biogas yield (SBY) of the HBT assays 1 and 2 based on volatile solid (VS) are shown for Sargassum muticum (SM) and wheat straw (WS) as well as for inoculum and the mixture of SM + WS. Contained VS and VS ratio (substrate to inoculum) as well as the gas production rate (GPR) for the last three days are given.

Parameter	Inoculum	SM	WS	SM + WS
SBY HBT 1 a (mL/gVS)	55.3 ± 3.06	117.2 ± 0.95	211.33 ± 19.9	175 ± 7.97
SBY HBT 2 b (mL/gVS)	42.3 ± 0.34	109.01 ± 5.43	243.63 ± 6.64	179.65 ± 4.69
C:N of substrate without inoculum	NA	14:1	71:1	38:1
Contained VS (g)	1.09	0.042	0.052	0.047
VS ratio of substrate to inoculum	-	0.038	0.047	0.043
GPR HBT 1 (%)	2.26%	0.70%	0.49%	0.58%
GPR HBT 2 (%)	1.33%	0.85%	0.36%	0.55%

Data obtained after 40 d of anaerobic digestion; NA = not analyzed. ^a ANOVA analysis of SBY mean values per measured time period of SM, WS, and SM + WS resulted in P = 4.19 × 10⁻⁸; F = 19.57; F_critical = 3.07. ^b ANOVA analysis of SBY mean values per measured time period of SM, WS, and SM + WS resulted in P = 1.8 × 10⁻¹⁴; F = 41.67; F_critical = 3.07.

). It can be seen that WS alone achieved a higher SBY than the SM + WS mixture. Nevertheless, if the SM + WS mixture in the HBTs is approx. 60:40, then the following mean values for a calculated SBY of the mixture based on the experimental results would result in 154 mL/g_VS for HBT 1 ((117 mL/g_VS × 0.6) + (211 mL/g_VS × 0.4)) and 162 mL/g_VS for HBT 2, respectively. When these calculated mean values are compared with the actual yields observed for the mixtures (175 mL/g_VS for HBT 1, 179 mL/g_VS for HBT 2), a positive effect for HBT 1 of 13% and for HBT 2 of 10.3% can be determined. Co-digestion of SM with WS can thus be recommended for S. muticum.

In addition, it can be assumed that an overall higher salinity was present in HBT 2 than in HBT 1, since the used inoculum from batch digester 8 already had an increased Na concentration compared to the mixture of the initial inocula used for HBT 1 and 2L-DE (Table 3, approx. 2 g/kg_DM for HBT 1 and 2L-DE compared to approx. 12.5 g/kg_DM for HBT 2). However, the SBY of the SM + WS co-digestion variant was approx. the same in both HBTs (Table 6), possibly indicating that the microbes can adapt to new substrates or that the experiment has not been in the inhibitory salt range.

Furthermore, the resulting SBY of the SM + WS mixture as determined in the HBTs was similar to the mean SBY of the untreated variant in the 2L-DE with 201.37 ± 30.38 mL/g_VS (Table 4) which allows for the validation of both test series. Small differences may have resulted from the different gas yield test systems, leaks, or reading inaccuracies. Furthermore, it can be seen that the degree of “overfeeding” (VS ratio of substrate to inoculum > 0.5, see recommendations by VDI 4630) in the 2L-DE did not negatively affect the results in the present case.

Since the SMY was not measured in the HBT assays, only the actual and stoichiometric SBY and the SBY of the 2L-DE can be compared. The actual SBY obtained for SM (Table 6) was approx. factor 9 lower than the theoretical value with an assumption of a 100% digestibility (theoretical: 1093 mL/kg_VS). For WS, the SBY was approx. factor 4.5 lower than the theoretical value determined with 957 mL/kg_VS. The mentioned values only refer to the untreated algae.

Vivekanand et al. [3] investigated the brown macroalgae Saccharina latissima, as well as the effects of thermal pretreatment and co-digestion with wheat straw, at different blending ratios on the resulting SBY. In their study, wheat straw was pretreated via steam explosion at 210 °C with a retention time of 10 min, which then led to an SMY of 98 mL/g_VS. The SMY of the stream-explosion-treated algae was lower than the yield of the untreated algae (223 mL/g_VS).

In the HBT assays in this study, only the SBY was measured, but if a methane concentration of 50% of the cumulated SBY is assumed, the SMY of WS would be roughly 106 mL/g_VS, which is within the range of the value found in Vivekanand et al. [3]. Nevertheless, there are other studies that found a significantly higher SMY for wheat straw. Theuretzbacher et al. [32] and Jackowiak et al. [33] found an SMY of 268 mL/g_VS for wheat straw after 41 d of anaerobic digestion and 240 mL/g_VS after 40 d of digestion, respectively. The SMY for WS as presented in this study was lower than in the literature. Thus, it could be concluded that the SMY of both SM and WS could also be higher in additional digestion experiments.

Furthermore, Vivekanand et al. [3] mention a positive mixing effect based on the better methane generation of WS and no inhibition of the algae via mixing the substrates was observed in their study. In addition, the blending ratio was found to have an influence on methane production and the highest SMY was found at a 70:30 (algae:wheat straw) blend. Also, Paul et al. [24] observed a positive blending effect when wheat and wild as well as cultivated algae Saccharina latissima are mixed.

Ayala-Mercado et al. [31] studied extrusion and steam-explosion pretreatment for Sargassum fluitans and S. natans. They referred to other studies that found an SMY from untreated Sargassum spp. of around 80–120 mL/g_VS and they observed positive effects on the SMY for both of their pretreatments. Their results showed that mechanical pretreatment (extrusion) increased methane production at the initial stage (day 4–23), after which, hydrothermal pretreatment had a slightly higher yield. They also suggest that mechanical pretreatment ruptures cells which increases the biodegradability of algae. However, better digestibility is not necessarily associated with higher methane production. Jard et al. [34] investigated the influence of the thermal pretreatment of the red alga Palmaria palmata on biodegradability and methane production. Their results showed that thermal pretreatment up to 160 °C leads to better solubilization, but not to increased methane production. In addition, a thermal pretreatment higher than 160 °C decreased degradability due to the formation of a group of phenolic macromolecules that inhibit anaerobic digestion.

Furthermore, the study of Montingelli et al. [35], which examined three algae of the Laminaria species, showed a negative effect of microwave treatment at 100 °C on methane production. Here, the pretreatment resulted in a 27% lower methane production compared to the untreated group. The results for the SMY as found in the 2L-DE of this study are similar to the before-mentioned studies, but the effects that occur during the pretreatment that ultimately lead to a differing SMY cannot be explained to a full extent. One inhibitor that microbes are not adapted to on a larger scale is salinity. A study on salt inhibition by the University of Hohenheim gives maximum limits of up to 15 g NaCl/kg_biomass [36]. Furthermore, Murphy et al. [11] mention that a Na ion level of 100–350 mg/L is needed for a healthy anaerobic digestion but an inhibitory effect can occur above 8 g/L. If only Na is considered, a concentration of 5–15 g/L can cause an inhibition during anaerobic digestion [37]. The imported salt concentration by SM could not be exactly determined but the element Na is used as a guiding element for salinity. For the untreated variant, the soaking and draining procedure of the algae resulted in 0.5 g of Na that was dissolved from the original substrate quantity in one single digester. This resulted in a Na content of 1.12 g per digester. Furthermore, one digester had a capacity of 2 L, resulting in a concentration of 0.56 g_Na/L. This is a lower amount than the mentioned inhibitory level as stated by Murphy et al. [11]. If the inhibitory range according to the study of the University of Hohenheim [36] is used, a Na concentration in the substrate of 0.018 g/kg_DM is calculated, which is not even close to the critical range. Thus, no salt inhibition is suggested during the experiments and the low SMY is likely attributed to other inhibitory processes.

Furthermore, it was found that Na concentrations in SM could be reduced by 31% mainly through re-hydrating and draining (although SM had already been rinsed with fresh water after collection). Through mechanical pretreatment, an additional 18% of the resulting Na concentration based on re-hydrated and rinsed SM (1.12 g_Na) was flushed out.

Moreover, it is also assumed that potentially missing trace elements are not responsible for the observed results. The untreated variant of SM + WS contains the most trace elements, and the highest amount of P was dissolved via the purely mechanical pretreatment, which also delivered the highest SMY. Nevertheless, fiber components are hardly degradable in anaerobic digestion but their structure, as mentioned in Borines et al. [21], might have been made (partially) accessible for the microbes via the mechanical pretreatment. This could explain why the mechanical variant performed better than the untreated variant. Moreover, it cannot be excluded that substances such as phenols may have formed during the thermal pretreatment which might have been the cause of the lower SMY even though heating was below 160 °C. Further studies on fiber components, their degradability, and phenolic inhibition of SM would be necessary for an accurate explanation.

By comparing the results of the batch experiment (Table 4) with the SMY yields of previously conducted studies with the algae genus Sargassum (

Table 7. A review of previously determined specific methane yields (SMYs) of Sargassum species based on volatile solids (VS) of the substrate without the influence of the inoculum.

Macroalgal Species	SMY	Reference
	mL/gVS
SMpressed + WS	101 ± 11	this study
S. muticum untreated	130 ± 1	[8]
S. muticum untreated	100 ± 50	[38]
S. muticum ensiled (whole)	110 ± 80
S. muticum chopped prior to ensiling	60 ± 10
S. muticum untreated	166–208	[9]
S. fluitans untreated	180	[39]
S. pteropleuron untreated	150
S. fluitans untreated	165 ± 8	[40]
S. pteropleuron untreated	145 ± 1

), it is concluded that the SMY measured in this study was not significantly improved, not even via the mechanical pretreatment of SM, which was the best variant in the digestion experiments of this study.

To present exact and general statements as to why a purely mechanical pretreatment of SM is advantageous compared to untreated SM or a thermo-mechanical pretreatment, further investigations and test series would be necessary. The analysis of the structural changes (caused by the pretreatments) or chemical reactions during the process might be an aspect that could complement the results of this study. In addition, the generated press waters and byproducts should be further investigated for possible applications.

Another aspect is to assess the overall economic viability, which includes several aspects. For instance, S. muticum and wheat straw are substrates that require no additional arable land. Costs for logistics are assumed to be low, at least in coastal regions, since S. muticum does not have to be cultivated (for instance, in comparison with energy crops). Furthermore, the expenditures for the mechanical pretreatment and process temperature as applied in this study have to be considered but they are already common in anaerobic digestion. The pretreatment, however, is only reasonable when achieving a higher SMY, which would also lead to larger revenues that would overcompensate expenditures for pretreatment.

An additional parameter that can be relevant for economic efficiency is salinity. In a continuous digestion process, with the assumption that there is no accumulation of certain elements, salinity in the digester will approx. correspond to the substrate mixture. All SM variants as analyzed in this study were uncritical with regard to the inhibition level as mentioned in Murphy et al. [11]. The addition of co-substrates such as WS could always reduce the salinity level and thus stabilize the anaerobic digestion process to achieve the best possible operating points.

In summary, the main driver of the concept’s economic viability remains the SMY in combination with an available market for renewable methane in the target region. Additional value can be achieved by creating ecological benefits, the upgrading of digestate as fertilizer, which contributes to a circular economy [41], or the material use of (undigested) fibers that are presumably highly concentrated in S. muticum. This was also mentioned in the context of another study, in which the low methane potential of SM reduced the economic attractiveness of a biorefinery [42]. Still, further research for now should focus on the cost-efficient but more effective pretreatment or digestion methods of Sargassum spp. to increase the SMY.

The necessity to valorize different, available algae such as SM is also discussed in the recent literature that deals with topics other than anaerobic digestion. For instance, the production of pharmaceutical or cosmeceutical products [43], bioactive compounds, or food [44] to tackle the overexploitation of land areas [45] is discussed in the context of SM.

4. Conclusions

Sargassum muticum has been studied with regard to its anaerobic digestion performance in different BMP test systems. It can be concluded that the co-digestion of SM with wheat straw (WS) has positive effects on the SBY and SMY. Through the co-digestion of SM with WS, the C:N ratio was adjusted, which could be a possible explanation for a better anaerobic digestion performance. Moreover, it was shown that a softer pretreatment method like a mechanical pretreatment (pressing/extruding) has a positive effect on the resulting SBY and SMY, especially when compared to thermo-mechanical pretreatment in which the SMY can even be decreased. Nevertheless, the determined SMY of S. muticum (approx. 100 L/kg_VS) remains relatively low compared to other biogas substrates such as maize silage or rapeseed (both typically between 300 and 400 L/kg_VS [46,47]), which makes S. muticum less attractive for energy production.

However, it would be important to utilize S. muticum economically since it is an invasive alga that is already available in large amounts. It could be an aspect worth investigating to use S. muticum or its digestates via other utilization approaches. The presumably high fiber content in S. muticum could allow for the generation of sustainable fibers for material use. Thus, on top of its utilization in anaerobic digestion, S. muticum could be relevant for not only generating energy but also products for material use, which calls for further research. Nevertheless, feasibility studies to evaluate the benefits and expenditures caused by substrate pretreatment should complement the results of this work.