Efficient reuse of Sargassum spp. biomass and organic fraction of municipal solid waste by anaerobic co-digestion in the Dominican Republic: Evaluation of biochemical methanogenic potential and reaction rates

Anaerobic digestion (AD) is a potential solution to valorize invasive pelagic Sargassum spp. Sargassum spp. (SP) biomass is characterized by a low carbon/nitrogen (C/N) ratio, which, in addition to the presence of indigestible fiber, sulfide, salt, ash


Introduction
Sargassum spp. is a genus of macroalga, phylum Heterokontophyta, belonging to the Phaeophyceae class.It gives its name to the Sargasso Sea, where these algae are particularly widespread.The increasingly abundant presence of Sargassum spp. is reflected in negative consequences at an environmental and economic level.One of the strategies for the valorization of Sargassum spp. is to produce biogas via anaerobic digestion (AD).
vapor [2] .It is useful as an alternative to dispose the organic waste and to produce green energy.The AD process is widely used to treat organic waste [2] , such as sludge [3] , agricultural production waste [4][5][6] , waste water [7] , macroalgae [8][9][10] , in order to produce renewable energy.Waste biomass co-digestion represents an emerging technology to improve AD performance.
Researchers' interest in energy production from macroalgae has been growing recently.Sargassum spp. is the object of numerous studies.The presence of this brown macroalga has grown exponentially since 2011 from western Africa to the Gulf of Mexico, known as the Great Atlantic Sargassum Belt (GASB) [11] .Sargassum spp. is invading the beaches of South Africa, the Gulf of Mexico, the Atlantic Ocean, and the Caribbean causing various problems for local communities [12] .The presence of this biomass on beaches leads to adverse effects on tourism and fishing.Furthermore, its decomposition produces hydrogen sulphide (H2S), a toxic gas with a bad smell [13] .Therefore, the recovery of this waste biomass arouses considerable interest since it would not only allow production of renewable energy but also solve the problems related to its abundant presence.Although the applications of Sargassum spp.are not limited to AD biogas production alone, this application is the most promising.However, there are insufficient studies on Sargassum spp.seasonally and regionally-dependent characterization and biochemical conversion to support significant advances toward finding solutions, The AD process of Sargassum spp.chiefly breaks down the cellulose fraction with yields lower than 50% in terms of biochemical methane potential (BMP) compared to theoretical.Low yields are attributed to the lignocellulose barrier, a low carbon/nitrogen (C/N) ratio, indigestible fiber, sulfide, salt, ash, and polyphenol content [12,14] .
To increase yields, Sargassum spp.must be subjected to pre-treatment.The pretreatments break down the lignin barrier [12] and increase accessibility to cellulose for downstream hydrolysis into glucose and towards methanogenesis.However, they also impact the overall energy balance as they increase the required energy to carry out the entire process [15] .Anaerobic co-digestion with organic waste biomass represents a valid and cost-efficient method to increase yields without any pretreatment and it allows and environmentally sustainable exploitation of these renewable energy sources, otherwise landfilled.The organic waste biomass increases the content of lipids, redistributes metal elements and raises the buffering capacity of the digester thus boosting the digestion performance [15] .
Recent literature has explored pretreatment techniques on pelagic Sargassum spp.followed by codigestion with the organic fraction of municipal solid waste (OFMSW).They reported that the maximum cumulative biomethane yields, equal to 293 NmLg −1 VS, was obtained by co-digestion of Sargassum spp. with OFMSW at a 25:75 weight ratio, after mechanical pre-treatment (size reduction) and heat-treatment at 353.15 K for 15 h of Sargassum spp., followed by hydrothermal pre-treatment pf SP and OFMSW in a pressurized batch reactor operating at 30 bars under N2 gas at temperature 413.15K for 30 min.and at stirring speed of 300 rpm [16] .
Oliveira et al. conducted a co-digestion study of Sargassum spp. with glycerol and waste frying oil.The co-digestion with glycerol and waste frying oil increased the BMP by 56% and 46%, respectively [17] .Rivera-Herná ndez et al. conducted a study in Mexico on the synergistic effect of the co-digestion of pig manure (PM) and Sargassum spp.(S) by testing five different ratios (100S-0PM, 65S-35PM, 50S-50PM, 30S-70PM, and 0S-100PM).The highest BMP of 441.47 mLg −1 VS was obtained in 50S-50PM treatment [18] .The arrival of Sargassum spp. in the Caribbean shores of the Dominican Republic has reached unsustainable levels.Even though efforts towards valorization and remediation exist across all levels, there are insufficient studies on Sargassum spp.characterization and biochemical conversion to support significant advances toward finding solutions.
The objective of this work is the valorization of Sargassum spp.species from the shores and beaches of the Dominican Republic via anaerobic digestion (mono-digestion and co-digestion) with OFMSW sourced from the area, , thus promoting the sustainability of AD implementation.From this point of view, this work is aimed to provide the optimized SP to OFMSW ratio in view of a potential year-round scale-up.During the months of non-accumulation of Sargassum spp. a plant would produce biogas from OFMSW only, and during the period of Sargassum spp.accumulation, it would work with a Sargassum spp.-OFMSW mix.

Sample preparation
A sample of the macroalgae Sargassum spp. was provided by the Punta Cana Foundation group.Samples were washed with deionized water.Excess water was removed with blotting paper.Samples were air-dried for several days until constant weight was achieved.After drying, the Sargassum ssp.samples were subjected to mechanical pre-treatment using a Philips-ProBlend Tech (Milan, Italy) mixer for 1 min at maximum speed.
The OFMSW sample was obtained from door-to-door municipal solid waste collection and was also subjected to the same mechanical pre-treatment.
The experiment was modeled by a modified experimental design methodology [19,17] .
The Sargassum spp.OFMSW organic matrix was prepared by mixing the previously prepared samples, according to Table 1.

Inoculum preparation
Inoculum was obtained by mixing water with cow manure (ratio 1:1).After being prepared it was kept at 384.15 K, a mesophilic temperature used subsequently for AD of the biomass, to acclimate it.After 30 days the volume of biogas produced by the inoculum was stable [20] .
Furthermore, for the sample of Sargassum spp. the content of metals and metalloids was determined using a Microwave Digestion System following the methodology used in a previous study [20] .
The content of metals and metalloids of the analyzed samples, expressed as mg kg −1 S.S., is calculated according to the following Equation 1 where B is the concentration (mg L −1 ) expressed by the ICP-MS analysis, V (mL) is the volume of the solution obtained from the mineralization and brought to volume to 50 mL, m the mass of the mineralized sample.

Determination of the experimental and theoretical methane potential
The experimental Biochemical Methane Potential (BMPex) was determined with the Automatic Potential System Test II (AMPTS-II®) reactor system manufactured by BPC instruments (Lund, Sweden).The tests were conducted in duplicate.Operating conditions: inoculum/substrate ratio equal to 3, mesophilic temperature conditions at 384.15 K [20] .
The theoretical methane potential was calculated following the BMPthCOD model [22] (Equation 2), based on the Chemical Oxygen Demand (COD) layer in the substrate.
where BMPthCOD is the theoretical methane production from COD (LCH4g −1 VS), COD is the chemical oxygen demand, nCH4 are the amount of molecular methane (mol), R is the gas constant (82 atm mLmolK −1 ), T the temperature of the reactor (310 K), p is the atmospheric pressure (1 atm) e VS the volatile solid of the substrate (g).

Kinetics of methane production
The kinetics of methane production was evaluated with two kinetic models: first order kinetic (Equation 3) [22] and Modified Gompertz model (Equation 4) [22] .
) where y(t) presents the biogas product during the AD process (NmLg −1 VS), A represents the amount of biogas that should be produced (NmLg , k represents the reciprocal value of time when y(t) reaches the value of 0.632A, u represents the daily amount of biogas (NmLg −1 day −1 VS), e it is a constant value (2.718282), m represents the lag phase period (days), and t is the time of AD process (days).

Sargassum spp. and OFMSW characterization
The chemical-physical composition of the samples of Sargassum spp.and OFMSW is reported in Table 2.In the sample of Sargassum spp.several metallics and metalloids are present.Some of them are present in negligible quantities.The contents of metals and metalloids are reported for those with content greater than 1%: Na 1.22%, K 3.35%, Ca 1.92%.The characterization of the prepared samples is reported in Table 3.In our previous study the chemical composition of Sargassum spp. was analyzed in detail, which varies according to geographical location and seasons.The ranges of lipid, protein, carbohydrates and ash content for the Sargassum spp. is 0.6%-2.7%,5.8%-14.1%,13.4%-46.1%,24.6%-76.4% respectively.
The content of VS (%), TS (%), ash (%) and moisture (%) of the samples under analysis are shown in Table 3. OFMSW sample (S1) was characterized by a low ash content while sample Sargassum spp.sample (S10) shows a much higher ash content that can be attributed to the bioaccumulation of minerals (e.g.Na, Ca, K, Mg) and trace elements (e.g.Fe, Zn, Ni, Cu) from the surrounding seawater [14] .The volatile solids content of OFMSW samples (S1) and Sargassum spp.sample (S10) was almost equal.The values of VS, TS, moisture and ash for the OFMSW sample are in line with what has been reported in other studies [23][24][25][26] .As regards the sample of Sargassum spp., since it is a very heterogeneous biomass, the content of VS (%), TS (%), ash (%) and moisture (%) varies depending on the collection site and the period [12] .The ash and moisture contents in the samples increase with the increase of the percentage of Sargassum spp.present in the sample while the moisture content with the increase of the percentage of OFMSW present in the sample.It is worth noting that the VS/TS ratios of PS and OFMSW are 0.49 and 0.95 respectively.They highlight that the latter contains more readily digestible compounds.The finding can be attributed to the high amount of total indigestible fiber content in SP and higher lipid/protein fraction in OFMSW.
The C/N ratios of PS and OFMSW are 8 ± 0.12 and 28 ± 015, respectively.While the C/N ratios of OFMSW is within the suggested optimal range of 20:1 to 30:1 for stable bio-digester performance [27] , the low C/N ratio of PS can lead to the formation of ammonium ions that increase the pH in the digester negatively affecting the methanogens bacteria.

Biochemical methane potential test
The BMPex evaluation test lasted 30 days.However, only the sample of Sargassum spp.(S10) took 30 days to reach the stationary state while all other samples reached it in just 10 days.The average cumulative values of BMP obtained after 10 days are shown in Table 4.The maximum total biomethane yield of 436.71 ± 29.33 NmLg −1 VS was achieved with the OFMSW sample (S1), in line with what has been found in other studies [27,28] .The minimum total biogas yield of 79.68 ± 2.38 NmLg −1 VS was achieved with the Sargassum spp.sample (S10) sample.Intermediate values were obtained for samples composed of a mix of Sargassum spp.and OFMSW at different percentages.It is possible to observe that the yield decreases with increasing amount of Sargassum spp.In fact, increasing the concentration of OFMSW also increases the C/N ratio, therefore the results are in line with what was expected.Thompson et al. [16] analyzed three different samples A1 (75% Sargassum spp.and 25% OFMSW), A2 (50% Sargassum spp.and 50% OFMSW) and A3 (25% Sargassum spp.and 75% % OFMSW) for 21 days at 35 ℃ and identified the optimal mix in the A3.The results can be compared with those obtained for samples S4-S6-S8 in the present investigation.The yield of sample S4 is equal to 345.64 ± 35.41 NmLg −1 VS after 10 days while the yield of sample A3 is equal to 201.67 ± 6.36 NmLg −1 VS.The yield of sample S6 is equal to 264.83 ± 16.36 NmLg −1 VS while the yield of sample A2 is equal to 182.33 ± 2.61 NmLg −1 VS.The yield of sample S8 is equal to 167.66 ± 11 NmLg −1 VS the yield of sample A1 is equal to 97.46 ± 1 NmLg −1 VS.In all three cases the yield obtained in the present investigation is higher.The results can be ascribed to several factors such the different operating condition and the different composition of OFMSW.Furthermore, the fact that the Sargassum spp. was collected in a different region and period can affect the overall results.However, also in our case, taking into account only the S2-S4-S6 samples, the highest yield is obtained for the 25:75 Sargassum ssp.-OFMSW sample.The Figure 1 shows the average cumulative volume of biomethane (NmLg −1 VS) produced during anaerobic digestion of the samples.From the curves it is possible to note that the Sargassum spp.sample (S10) has a growing trend.Indeed, for this sample a stable production level was reached only after 30 days.The sample of OFMSW alone instead has a higher production speed and therefore the AD process reaches a stable trend in 10 days.

Determination of the theoretical potential
The data reported in Table 5 show the yield of the samples compared to the theoretical yield.It is important to underline that for Sargassum spp. the experimental yield is always lower than the theoretical yield.Analyzing the data obtained, it is possible to observe that the yield increases with the increase of the percentage of OFMSW present in the samples.A comparison between the values of BMPth and BMPex shows that for Sargassum spp.sample (S10) it is very far from the theoretical yield and that by decreasing the percentage of Sargassum spp.present in the samples the two values get closer.Actually, the Sargassum spp.sample (S10) reaches only the 14.41% of the theoretical yield.When the sample of Sargassum spp.OFMSW is added, the value of the experimental yield increases.In this way the gap between experimental yield and theoretical yield is reduced.Thus, the sample containing 10% Sargassum spp.and 90% OFMSW (S2) reaches 73.07% of the theoretical yield.The sample of OFMSW (S1) reaches 84% of the theoretical yield.This indicates that the OFMSW sample is more easily degradable than Sargassum spp.

Kinetic production
First order kinetic model and Modified Gompertz model were used in order to find the kinetic parameters.Figure 2 shows the curves.From the curves it is evident that the AD process develops in three phases.The first phase, called lag phase, represents the period necessary for the first quantity of biogas to be produced.The second phase coincides with exponential growth while the third phase represents the stationary phase.
The parameters estimated using the two fitted kinetic are reported in Table 6 and Table 7.The data obtained from the fitting indicate that the Gompertz model fits the data better than the First order kinetic model.For all samples the values of A obtained using the Gompertz model deviate slightly from the experimental value, the values obtained for samples S1, S2, S3, S4, S5, S6, S7, S8, S9, deviate by 26.98 %, 0.38%, 0.07%, 4.44%, 1.42%, 0.56%, 3.15%, 5.44%.13.90%, respectively from the experimental values.The m (day) parameter indicates the delay time, for all the samples it is less than one day.The parameter u (NmL g −1

VSday
−1 ) indicates the daily production of biomethane.The value of R 2 indicates how well the model fits the data.Furthermore, for sample S10 in Figure 3 the fitting is reported not only at 10 days but also at 30 since the biogas production of this sample reaches a stable value after 30 days.The parameters estimated using the two fitted kinetic for the Sargassum spp.sample (S10) after 30 days are reported in Table 8.

Figure 2 .
Experimental data fitted by the first order kinetic model and modified Gompertz model.

Figure 3 .
Figure 3. Experimental data fitted by the first order kinetic model and modified Gompertz model for the sample S10 after 10 days and 30 days of anaerobic digestion process.

Table 3 .
Characterization of the prepared samples.

Table 6 .
Kinetic parameters of the first kinetic model.

Table 7 .
Kinetic parameters of the modified Gompertz model.

Table 8 .
Kinetic parameters of the First kinetic model and Gompertz model for sample S10 after 30 days.