Efficiency of Fe3O4 Nanoparticles with Different Pretreatments for Enhancing Biogas Yield of Macroalgae Ulva intestinalis Linnaeus

In this work, different pretreatment methods for algae proved to be very effective in improving cell wall dissociation for biogas production. In this study, the Ulva intestinalis Linnaeus (U. intestinalis) has been exposed to individual pretreatments of (ultrasonic, ozone, microwave, and green synthesized Fe3O4) and in a combination of the first three mentioned pretreatments methods with magnetite (Fe3O4) NPs, (ultrasonic-Fe3O4, ozone-Fe3O4 and microwave-Fe3O4) in different treatment times. Moreover, the green synthesized Fe3O4 NPs has been confirmed by FTIR, TEM, XRD, SEM, EDEX, PSA and BET. The maximum biogas production of 179 and 206 mL/g VS have been attained when U. intestinalis has been treated with ultrasonic only and when combined microwave with Fe3O4 respectively, where sediment were used as inoculum in all pretreatments. From the obtained results, green Fe3O4 NPs enhanced the microwave (MW) treatment to produce a higher biogas yield (206 mL/g VS) when compared with individual MW (84 mL/g VS). The modified Gompertz model (R2 = 0.996 was appropriate model to match the calculated biogas production and could be used more practically to distinguish the kinetics of the anaerobic digestion (AD) period. The assessment of XRD, SEM and FTIR discovered the influence of different treatment techniques on the cell wall structure of U. intestinalis.


Introduction
Due to their high polysaccharide content and low lignin concentration, macroalgae (seaweeds) have tremendous potential as a feedstock for bioenergy production [1,2]. Large, multicellular sea organisms abound in nature, accounting for over half of the world's biomass population [3,4]. Seaweeds fix atmospheric CO 2 for photosynthesis and can multiply quickly, due to a 4-fold higher photosynthetic efficiency than terrestrial biomass [5].
In the year 2000, 11.4 million wet tones of seaweed were collected globally [6]. Pretreatment strategies have been investigated to solve the problem of low CH 4 productivity. These approaches improve organic matter bioavailability for microbial hydrolysis, reducing hydraulic retention time (HRT) and enhancing biogas production [7,8].
Pretreatment is a common strategy for speeding up the AD process and increasing biomethane production by making previously inaccessible substrates accessible to microorganisms and speeding up the substrate conversion process. The effects of a pretreatment on a particular substrate depend not only on the pretreatment mechanism but also on the characteristics of the substrate [9][10][11].
Because employing many mechanisms and combined techniques that are usually more efficient than methods that use only one tool is also more complex. For example, Rafique et al. [28] studied the effects of thermal, chemical, and thermochemical pretreatment on dewatered pig manure. At 70 • C, high concentrations of lime (5%) exhibited the most significant increase in gas output, far better than lime alone or heat alone. During batch AD experiments, there was a 78% increase in biogas. The importance of this research comes from the necessity to combine the nanoparticles treatment with different thermal, chemical and physical treatments. This works into two distinct pathways: first, working on enhancing the enzymatic activities and the second work on the degradation of the substrate's cell wall, which increases the ability of biogas production. Moreover, the evaluation of the impact of different treatments and the cell degradation will also be investigated using FTIR, TGA, SEM, and XRD techniques.
In the present study, we have analyzed the impact of two process parameters on biogas production from the macroalgae U. intestinalis. First, this work examined how thermal, physical and chemical treatment and nanoparticles affect U. intestinalis to break down biomass and their effect on biogas productivity. Second, the combination of U. intestinalis pre-treated with iron NPs for biogas production was evaluated to verify whether different processing methods could affect seaweed biogas production. This is the first study that describes the effect of a mixture of three different treatments, either singly or in conjunction with magnetite NPs, when employing sediments as a source of anaerobic bacteria.

Collection of Green Algae U. intestinalis
U. intestinalis, a fresh marine green algae, was hand-collected off the Mediterranean shore of Alexandria, Egypt. The biomass was cleaned several times with seawater, tap water, and distilled water before being used. The clean algae was sun-dried for several days before being oven-dried for 24 h at 50 • C. The biomass was then grinding to obtain a fine and homogeneous powder. The dried samples were milled to a size of about 0.5 mm using (Fritsch, Pulverisette 2, Idar Oberstein, Germany) for 5 min, and the milled seaweed samples were placed in plastic bags and kept at room temperature until further use.

Chemical Analysis of Algae Powder
The dry matter has been calculated. By ashing the ground dried samples overnight in a muffle furnace at 550 • C, the ash content was measured. The elemental analyzer was used to calculate C, H, N and S (elemental analysis Vario Micro Cube, Langen selbode, Germany).

Ozonation Pretreatment of U. intestinalis
Using a 0.2 L cylindrical glass containing 150 mL of U. intestinalis algal suspension as the working volume, ozonation pretreatments were carried out at a flow rate of 8.3 mg O 3 min −1 , O 3 was guided into the column via a porous glass sprinkler. Using an O 3 generator, O 3 was produced (N 1668 a power: 18 W, Vol AC 220 V/50 HZ). All ozonation experiments were performed at pH 8, since when the pH is higher than 7.0, the O 3 decomposition rate increases dramatically at room temperature (23 ± 2 • C) due to hydroxyl radical formation and three curing times (t) (10, 15, and 30 min) were checked [12,22].

Sonication (US) Pretreatment of U. intestinalis
Using a 0.2 L cylindrical glass containing 150 mL of U. intestinalis algal suspension as the working volume, US pretreatments were carried out at pulse 99 and amplitude 99%. Using an US homogenizers, the pulse sonication effect was produced BY Model CY-500-US Homogenizers for three curing times (t) (10, 15, and 30 min). The ultrasonic frequency was 20 kHz. The horn is made of titanium alloy with variable power output rates to vary the effect of the ultrasonic application. The ultrasound probe was made with 1/4 inch titanium alloy (5.6 mm and 60 mm height) [16].

Microwave Pretreatment of U. intestinalis
Using a 0.25 L polytetrafluoroethylene (PTFE) or Teflon lined hydrothermal autoclave reactor containing 150 mL of U. intestinalis algal suspension as the working volume. MW pretreatments were carried out at 1100 watts for two curing times (t) (2, and 4 min) by sharp watts MW system [16].

Green Synthesis of Fe 3 O 4 Nanoparticles
The magnetite (Fe 3 O 4 ) NPs were synthesized according to the following method: where 10 g of the dried U. intestinalis were refluxed in 100 mL double-distilled water (DDW) for 3 h. Then, the refluxed solution was filtered, and the filtrate was used as the reducing agent. FeCl 3 ·6H 2 O and FeSO 4 ·7H 2 O in a 2:1 M ratio were added and sonicated for 10 min. After that, the solution was heated to 80 • C for 10 min while magnetically stirring. Then, at 80 • C and steady stirring, 5 mL U. intestinalis extract and 20 mL 1 M NH 4 OH were added drop by drop. The obtained colloidal suspensions were then centrifuged, washed repeatedly with ethanol and then dried at 70 • C for 24 h. The final product was calcined at 550 • C to obtain the Fe 3 O 4 NPs [29].

Fe 3 O 4 NPs Pretreatment of U. intestinalis
A stock solution of the Fe 3 O 4 NPs at a concentration of 1 g/L was prepared by dispersing the nanopowder into Milli Q water (conductivity of 18.2 MU/cm at 25 • C). For shock loading, the generated Fe 3 O 4 NPs solution was diluted to 5, 10, and 20 mg/L in the current study. All of the tests were carried out in duplicate and the T-test in Microsoft Excel was used to calculate the significant difference between the studies.

Inoculum and Substrates Preparation
Microbial seed was obtained from marine sediment collected from El-Mex pump stations (Lat: 31.12486111 and Long: 29.87916667) at Alexandria, Egypt. As proposed by Santegoeds et al. [30] and Do Nascimento et al. [31], 300 g were introduced to 1200 mL sterilized seawater (pH 7) enriched with volatile fatty acids (VFA) mixture and supplemented with nutrients. The inoculum mixture was sealed after removing O 2 with N 2 gas.

Biogas Tests
Laboratory tests were conducted on reactors in similar digesters of cylindrical syringes [27,32,33]. The syringes are reversed directly onto the reactor lid [34,35]. A plastic syringe was used to sample the fuel that was equipped with a three-way valve and reinjected into the waste. In all tests, 100 mL glass syringes were applied. As feedstock, 1.5 g of milled U. intestinalis (dried weight) was used. In each syringe, 20 g (wet weight) of sediment was applied to the untreated and treated U. intestinalis. For 10 min, the working volume was flushed with N 2 . For each anaerobic degradation set-up, three replicates were performed. Until no apparent methane was produced, the inoculum was pre-incubated for three days. At 37 • C with continuous shaking at 150 rpm, the digesters were incubated. Table 1 offers an overview of the substrates used in batch experiments to estimate the U. intestinalis biogas yield.

Characterization and Measurement
The following procedures were used to characterize Fe 3 O 4 NPs, and U. intestinalis samples before and after pretreatments with US, O 3 , and MW: Model V-100 VERTEX70, Germany, Fourier transform infrared (FTIR) spectroscopy (platinum ATR) in the wavenumber range (400-4000 cm −1 ), X-ray diffractograms (XRD) were obtained with a Bruker Meas Srv (D2-208219)/D2-2082019 diffractometer operating at 30 kV, 10 mA, and a Cu tube (=1.54) with a 2Theta (2θ) range of 0 to 100 • . For both Fe 3 O 4 NPs and U. intestinalis, the surface structure was examined using a JEOL 6360LA scan electron microscopy (SEM). TERIOS Universal V4.5A TA Instruments (New Castle, DE, USA) performed thermogravimetric analysis (TGA) of the impregnated sample for U. intestinalis before and after US, O 3 and MW pretreatments. The prepared green nanostructure Fe 3 O 4 was characterized individually by Raman (the sample was exposed to this beam for 1 s at 10 mW power with aperture 25 × 1000 mm, three distinct points were measured and displacement occurred between 100 and 1400 cm −1 ), SEM with energy dispersive X-ray spectroscopy (EDX) detector (used to analyze elemental composition of substances), transmission electron microscope (TEM) (JEOL, Model JSM 6360LA, Tokyo, Japan), PSA (The Malvern Mastersizer 3000 is a compact optical instrument that employs laser diffraction to assess particle size distribution), mean pore diameter, and specific surface area were measured on BELSORP (Mini II, BEL Japan Inc., Osaka, Japan) using the BET method (Brunauer-Emmett-Teller) [36].

Kinetics Study and Statistical Analysis
Numerous researchers have used the nonlinear regression models, and the modified Gompertz Equation (1) was applied to determine the cumulative biogas production [37,38]. The model mainly determines the lag phase, biomethane potential, and the max biogas production rate. The biogas production data and the kinetic parameters were defined under widely recognized Equation (1) [37][38][39].
where Pb is the maximum biogas capacity of the substrate (L/g VS added), t is the duration (day), Rm is the maximum bio-gas rate, and e is 2.7183, where M is the biogas yield (L/g VS added) over time t (days). To compare the accuracy of the researched models estimated using SPSS 20, Origin 2020b, and Excel 2010 methodologies, the coefficient of determination (R 2 ) and root mean square error (RMSE) for both models were obtained. The standard deviation is interpreted as the RMSE, with a lower RMSE implying a better match between predicted and measured values [37,39].
where PVi is the estimated biogas volume value, MVi is the measured biogas volume value, and n is the number of measurements.

Characterization of Green Fe 3 O 4 NPs
FT-IR spectroscopy was used to investigate the functionalization of green generated magnetite nanoparticles. In the FT-IR spectra of Fe 3 O 4 NPs in Figure 1, the absorption peak at 530.36 cm −1 is strong. This also shows that the magnetic core is present, as nanoparticles of bare magnetite seem magnetized. The absorption bands 1469.59, 1645.09, and 2333.6 are associated with C-H, C=O, and aromatic components. These bands originate from the extraction of algae. At 3396.25 cm −1 , the stretching vibration of OH was captured by the band. As a result of hydrolysis on the surface of Fe 3 O 4 , the nanoparticles were hydrated (Fe(OH) 2 , Fe(OH) 3 , and FeOOH) [38][39][40][41]. Therefore, the absorption peak at 436 cm −1 indicated that present goethite might be formed by oxidation of Fe(OH) 2 according to the below reaction in atmosphere in excess NaOH (Equation (3)):

Raman Spectroscopy
Raman spectroscopy was utilized to identify the iron oxide core (magnetite) type. The Raman spectrum peaks of magnetite were investigated in Figure 2, where five vibrational modes are 212 (T2g(1)), 271 (Eg), 398 (T2g(2)), 493 (T2g(3)) and 659 cm −1 (A1g) for the magnetite [42][43][44]. Raman spectrum includes a strong peak located at 385 cm −1 . Other less intense peaks at 584 and 685 cm −1 indicated goethite (FeOOH), and the peak vibrated at 1069 cm −1 was related to the organic compound of the capping agent. According to Testa-Anta [45], most metal-oxygen lattice vibrations occur below 750 cm −1 , the main vibrations of organic molecules occur above 1000 cm −1 .  (Figure 3). This demonstrates that the Fe 3 O 4 nanoparticles are generated in this work. The small peak visible at 2θ = 24.17 • is attributed to the goethite structure (FeOOH) corresponding to the (110) plane. It is not easy to differentiate these structures even if both phases exhibit high crystallinity. However, some authors report that in the XRD pattern associated with the goethite phase, there exist two additional peaks located at 24.17 • and 33.12 • (211) and (104) [35].

Scanning Electron Microscopy (SEM)
The creation of large agglomerates of nanoscale particles can be attributed to the expansion through coalescence of nuclei, resulting in particles that tend to cluster toward a lower energy state free, by reducing interfaces with the environment, as shown in the SEM image ( Figure 4). The elemental composition of Fe 3 O 4 is determined by EDX. Table 2 showed that the iron and oxygen contents are 81.13 and 18.87 mass%, respectively. It can be seen that the oxygen content increases with the presence of goethite (FeOOH) indicating the presence of magnetite as the host material with a small portion of goethite.

Transmission Electron Microscopy (TEM)
TEM micrograph ( Figure 5) showed spherical agglomerated particles; agglomeration might be due to solvation and capping of nanoparticles by algal extract [33]. The particle sizes are in the range 5.6-16.8 nm.   3.1.6. Particle Size Analyzer (PSA) and BET Analysis of the Surface Area Figure 6 depicts the PSA-defined particle size distribution for Fe 3 O 4 NPs. The Fe 3 O 4 nanoparticles have a uniform particle size, as evidenced by the detection of Fe 3 O 4 particles in the range of 6 to 8 nm using a 10 • test angle and another range of particle size from roughly 200 to 257 nm using a 90 • test angle. The green Fe 3 O 4 NPs' uniformity and homogeneity may have a good impact on biogas generation. The BET analysis of green Fe 3 O 4 shows that the synthesized magnetite nanoparticles' surface area and average pore size were 37.85 m 2 /g and 9.56 nm, Table 3.   Figure 7 depicts the FTIR spectra of U. intestinalis biomass before and after pretreatment with US, O 3 , and MW, which show relative peaks at different wavenumbers. This is because, while only water is utilized during pretreatment, the above-mentioned pretreatments do not add new chemical groups to the solid fraction of seaweed following pretreatment. It reveals that there are two absorption peaks in the hydrogen bonding area ranging from 2927 to 3729 cm −1 . The stretching vibrations of hydrogen-bond O-H and N-H groups are assigned to the primary peak at 3267 cm −1 , showing phenolic and alcoholic chemicals, carbohydrates, and proteins in the U. intestinalis biomass. The vibration of the C-H group in the polysaccharides in the U. intestinalis biomass is responsible for the soft peak at 2964 cm −1 . The C=O group of amides, which arises due to proteins, is ascribed to the peak at 1641 cm −1 . The peak around 1429 cm −1 could be attributed to the aromatic structure's C-C stretching vibration. The peak at 1083 cm −1 is typical of aliphatic amines' C-N and C-O-C stretching vibrations, indicating the presence of proteins and polysaccharides [46,47]. The FTIR spectra show that raw seaweed displayed high stretching vibration peaks corresponding to the O-H and N-H groups (3454 cm −1 ). However, these absorption strengths decreased in the processed seaweed, suggesting the decomposition of carbohydrates and proteins. The vibration peaks corresponding to the C-H and C-C groups (2964 and 1429 cm −1 ) similarly dropped in a similar manner, which was related to hydrolysis of polysaccharide components after pretreatment with US, O 3 , and MW. The decrease in C=O and C-N vibration peaks (1641 and 1083 cm −1 ) demonstrated that the proteins in the treated seaweed had degraded.

X-ray Diffraction (XRD)
The initial degree of crystallinity is a crucial element to assess the pretreatment process. The crystallinity of raw and pretreated U. intestinalis was studied using X-ray diffraction analysis (Figure 8). The crystallography showed that after US, O 3 and MW pretreatments, the peak strength of the raw U. intestinalis sample became sharper at 2, 20, 26, 27, and 30, Figure 8. These peaks tend to conform to crystalline cellulose after pretreatment. This may prove that the pretreated U. intestinalis crystallinity increased when pretreated with US, O 3 and MW.

Thermal Analysis (TGA)
According to the findings in Figure 9, the thermal degradation of algae samples occurred in three stages. In the first level, from 70 to 100 degrees Celsius, there was a weight loss, which can be explained by evaporation of the sample's moisture content [48] or some light volatile materials [49][50][51][52]. On the other hand, the second level took place from 100 • C to a temperature of up to 400 • C. As a result of the significant deterioration process, consequential weight loss was noted at this point. Organic components of algae, such as carbohydrates, protein, and lipids, decompose and/or depolymerize, resulting in this loss. The mass loss of algae between 180-270 • C is due to carbohydrate decomposition, while protein degradation occurs between 320-450 • C, and the third stage linked to lignin decomposition occurs and restart from 500 • C [39]. In our study, the peak in range 626.85-826.85 • C may be due to the macroalgae's ability to remediate heavy metals by uptaking these metals inside their cells and remains in the U. intestinalis after pretreatments because they create cavities and blockage in the outer cell wall [48].

Surface Morphology, Scanning Electron Microscopy (SEM)
SEM evaluated the influence of the pretreatments on the structure of macroalgae. The morphology of macroalgae provided an alternative view, giving a better understanding of the effect of using US, O 3 and MW rather than untreated U. intestinalis, to better understand the effect of the pretreatments on the AD of the green macroalgae U. intestinalis. Un-treated U. intestinalis ( Figure 10) indicates a braided canvas with only a few broken fibers. At the same time, the pre-treated samples of U. intestinalis showed that it is possible to distinguish broken cell walls and disrupting their integrity. This suggests, therefore, that the pretreatments could harm the algae cell wall and create hydrodynamic cavities with a more superficial degradation and blockages, but the U. intestinalis treated with the US was better than O 3 and MW [23].

Chemical Compositions of U. intestinalis
As shown in Table 4, the VS content of the investigated U. intestinalis is 70.55%. On the other hand, by means of an elemental analyzer, the determination of the C and N material is detected and the measurement procedure is followed [50]. Table 4 shows a C/N ratio of about 9.60%. It is also worthy to mention that, the reported literature [53][54][55] indicated that the optimum C/N ratio is 16-19% for better methanogenic efficiency when considering hardly degradable complexes such as lignin [51]. However, this ratio is near the same ration for the present studied biomass U. intestinalis.

The Impact of Different Pretreatment Techniques on Anaerobic Digestion and Biogas Production
The experimental findings of biogas outputs were collected over a 42-day period, as shown in Figure 13. In the beginning, hydrolysis and fermentation were the main processes, and the biogas yield was relatively low. Following the initial anaerobic process that provided substantial biogas outputs in the first step, inactivity presumably due to the methanogens undergoing a metamorphic growth phase [56,57]. When the U. intestinalis was treated with US (pulse 99 and amplitude 99%), O 3 dose (8.3 mg O 3 min −1 VS) with time intervals of 10, 15 and 30 min and MW (1100 Wt) with time intervals of 2-4 min, the average biogas production yield was marginally increased compared to the biogas production yield obtained from the untreated U. intestinalis. The best biogas results production (206 mL/g VS) in this study was attained when the substrate was treated with MW in combination with Fe 3 O 4 NP with a concentration of 5 mg/L, Figure 13e. This biogas yield is more than two and a half times greater than of the individual MW treatment (84 mL/g VS). Figure 13a, displays the tested US treatment properties as duration (10, 15, and 30 min), demonstrating that US treatment can improve algal cell wall solubility, allowing for enhanced biogas production via anaerobic digestion or acceleration of the anaerobic process. The second better biogas production yield were from those with the shortest US time treatments. In particular, the highest biogas generation (179 mL/g VS) was observed for a US period of 10 min Figure 13a. These values were accepted as the optimal dose in US treatment before anaerobic digestion testing, where they are higher than those generated from untreated mixed anaerobic digestion. Moreover, the higher US time for 15 and 30 min has inhibitory effects on the biogas generation due to the development of less biodegradable by-products than the untreated substrates. Figure 13b,d demonstrates the effect of ozonation and different Fe 3 O 4 NPs concentration on the biogas production from U. intestinalis. The biogas yield was higher than individual MW treatment with biogas yield of (162 and 154 mL/g VS) for O 3 10 min and FE 3 O 4 5 mg/L treatments, respectively. As seen in Figure 12, the biogas output tests were completed when the regular production of biogas was <1% of the total production of most of the tests conducted.

Discussion
The usage of US in liquid solutions is based on monolithic cavitation, which has physical and chemical effects [58]. The physical impacts are caused by the collapse of cavitation bubbles, which results in an increased chemical modification due to the creation of free radicals [59]. Microbiological cells can be destroyed, and harmful chemical substances can be oxidized, as a result of these impacts [60,61]. Several studies [62][63][64] allude to the use of sonolysis to increase COD solubility and anaerobic biodegradability of sewage sludge prior to anaerobic digestion in the United States; they demonstrate that sonolysis can greatly improve COD solubility and anaerobic biodegradability of sewage sludge. Kim et al. [65,66] discovered that when US pretreated sludge was compared to untreated sludge, methane generation rose by 34%. Low-frequency degradation of surplus sludge has been demonstrated to be more efficient: mechanical effects increase particle solubility, ensuring the availability of higher amounts of readily digested organic materials in the liquid phase [66,67]. Despite the fact that US generation consumes energy, it has been observed that the US process modifies the organic matter structure, making it significantly more homogeneous and less sedimentary [58]: as a result, less energy (500 W-50 Hz) is required to improve particle solubility in the digesters. However, to determine the practical costs and benefits, additional, comprehensive research in a broader size is needed. In our work, the U. intestinalis treated with 10 min ultrasonic produce highest cumulative biogas production 179 mL/g VS, which means that ultrasonic pretreatment could promote the hydrolysis of carbohydrate polymers to reducing sugar.
Lower O 3 doses have significant positive effect on the production of biogas (p < 0.05). The ozonation time of 10 min produces a higher biogas yield with 162 mL/g VS for U. intestinalis combined with sediments. Because of the generation of less biodegradable by-products than untreated substrates, biogas quantities produced by O 3 pretreatment were found to be larger at 10 min than those generated by untreated mixed anaerobic digestion and for treated with O 3 durations of 15 and 30 min. According to these findings, compared to time 10 min, exposure to O 3 for longer than 10 min does not determine additional oxidation effects. From the previous results of different biotechnology fields, it is safe to assume that the promise of ozone can also be used in the anaerobic digestion process to enhance the ferment ability of the macroalgae biomass. Hassaan et al. [22] stated that the higher doses of O 3 (15 and 30 min) increased the biogas ability of the studied green algae Ulva lactuca, compared to untreated biomass studies when he uses manure and sludge as a source of bacteria. This variability could be attributed to the source of manure, wherein in our research, we have used sediment, which contains different media for AD. The evaluation of FTIR, TGA, SEM, and XRD in this study are in agreement with the results obtained by Hassaan et al. [22], which revealed the impact of O 3 on the structure of the algal cell wall and integrity breakage (Figure 10), which was thus established as the main contributor to improving the biogas production.
It is clear that the MW treatment only gives the lowest biogas yield among the studied treatment techniques with 84 mL/g VS after 2 min of treatment. Wu [16] stated that the Microwave pretreatment with a power of 700 W could improve biogas production in the range of 7.8-43.7% when applied for 1.5 min and in the range of 37.2-45.2% when applied for 3 min. However, several studies showed that microwave pretreatment had no or adverse impact on biogas production. The small biogas production through microwave pretreatment may be explained by the change in osmotic pressure and the output from solubilization of lignin through microwave pretreatment, which has detrimental effect on anaerobic bacteria [16]. On the other hand, when comparing the various treatments and the control, the results showed that MW pre-treatment in combination with Fe 3 O 4 NPs produced the best biogas yields. The amount of biogas produced by the MW pretreatment + Fe 3 O 4 NPs group was 206 mL/g against the control group. Multiple pretreatment combinations have been examined to improve biomass enzymatic hydrolysis and the corresponding BMP. They cannot be classified as mechanical, thermal, or chemical pretreatment because they involve a combination of methods. While combination pretreatments are more complicated than typical treatment techniques, they are more successful.
Finally, it is worse to mention that when U. intestinalis was treated with Fe 3 O 4 of 5 mg/L it gives 154 mL/g Vs biogas yield which is higher than MW treatment alone and the order of biogas production according to the used techniques can be arranged as the following: MW + Fe 3 O 4 > US > O 3 > Fe 3 O 4 > MW. It is also worth to mention that the little dosage of the treatment give higher biogas yield when we use sediment as manure and this behavior needs more investigation. Low dosage have a significant positive effect on the production of biogas (p < 0.05). When compared to untreated U. intestinalis, the improvement in combined Fe 3 O 4 with MW treated U. intestinalis and individually sonicated U. intestinalis is greater than other NP treatments, such as nano zero valiant iron (nZVI) and Fe 2 O 3 NPs with concentrations of 10 and 100 mg/g TSS, which produces more cumulative methane at 120 and 117 percent of total biogas yield, respectively [68].
However, addition of the magnetic Fe 3 O 4 NPs was found to improve biogas production more than untreated algae at 20 mg/L maximum concentration, which is consistent with Abdelsalam et al. [24], who concluded that magnetic NPs appeared to be non-toxic during long-term contact and only exhibited mild toxicity to bacteria at the initial stage. In contrast, our research found that adding 20 mg/L Fe 3 O 4 magnetic NPs to biogas production during starting and over the first 42 days of HRT increased bacterial activity. According to our findings, the best biogas productivity was produced utilizing 5 mg/L Fe 3 O 4 magnetic NPs in combination with MW treated macroalgae. These findings showed that Fe 3 O 4 magnetic nanoparticles improved anaerobic digestion, increasing biogas production and organic matter decomposition. The presence of Fe 2+ /Fe 3+ ions, which were injected into the reactor as nanoparticles and could be adsorbed as a growth ingredient for anaerobic microbes, increased performance [24].
The physiochemical properties confirm the presence of magnetite (Fe 3 O 4 ) and a small amount of goethite (FeOOH), where magnetite release of bioavailable ions (Fe 2+ and Fe 3+ ), which is known as an essential nutrient for microbial power generation. DNA replication [69] and key enzymes formation furthermore, increased concentration of acetate and butyrate which are known as energy favorable volatile fatty acids (VFAs) for the methane production phase [70], may consequently promote the AD process, as well as better effluent quality [71]. Therefore increase microbial abundance and activities of key enzymes or coenzymes. Furthermore, increased concentration of acetate and butyrate, known as energy favorable volatile fatty acids (VFAs) for the methane production phase [70], may consequently promote the AD process and better effluent quality. Serve as conduits for electrons, hence stimulate electron transfer between the bacterial and archaeal communities to reduce carbon dioxide (CO 2 ) to CH 4 [72]. While, the goethite (FeOOH) maybe act as capable of absorbing inhibitory compounds pollutant species, including a high abundance of ammonia, phosphorus and sulphate, together with excessive amounts of heavy metals in wastewater treatment rapidly and thoroughly via precipitation [73,74] and trap these compounds on their surface [75,76]. Generally, both iron oxides (magnetite and goethite) works as a pH buffer, thus stabilize the AD system [75,76]. Finally, all the publications concluded that the influences on the AD process nano-iron-additives were dosage-dependent. An excessive dosage of iron-based nanoparticles hindered the overall process resulting in reductions in biogas production. The results indicated that iron oxide NPs additives have a positive impact and improved biogas production by releasing two electrons due to oxidation to Fe 2+ under anaerobic conditions [77]. The electrons released by Fe can be consumed by inorganic CO 2 or acids and accelerated by the hydrogenation pathway and thus produce more CH 4 [77]. Where the highest specific biogas production (154 mL/g VS) for untreated algae with Fe 3 O 4 NPs of 20 mg/L.

Kinetic Study
The modified Gompertz equation has been shown in previous studies to be a standard model for biogas production from a simple organic substrate. The modified Gompertz model was used to fit the cumulative methane yields obtained from the anaerobic codigestion trials. Figure 13 shows a comparison of fitting results produced from the modified Gompertz model and experimental data. As observed in Table 5, the R 2 values were all greater than 0.9, indicating that the modified Gompertz model performed well in representing the cumulative process of biogas output. Table 5 summarizes the results of the kinetic investigation on gas production. It is reported that the Gompertz models matched well with the experimental findings. The late reaction and eventual microorganisms adaptation to the fluctuating atmosphere is expressed in the lag phase (λ) [37,46]. The modified Gompertz model have λ values of 0.0227 and 0.0287 days, for US 10 min and MW + Fe 3 O 4 , respectively. The calculated values for biogas generation are displayed against the observed values to assess the trustworthiness of the model findings in the tested model. The low RMSE (0.659) and (1.44) data indicate that modified Gompertz can dependably predict high bioactivity. Table 5 lists the statistical indicators (R 2 ). The higher R 2 (0.996 and 0.993) and lower RMSE values for the modified Gompertz model indicated a more acceptable kinetic model, according to Nguyen et al. [37]. The results showed that the experimental data could be fitted with the modified Gompertz model and could use the model to determine the cumulative biogas production, max biogas potential, maximum biogas production rate and lag time. It is also clear from Figure 13j the biogas data for the predicted and experimental is not fitted well, which is also confirmed by low R 2 (0.858). On the other hand, in Figure 13h, the biogas data for both predicted and experimental is fitted well and in a good arrangement, which is confirmed by high R 2 (0.992).

Conclusions
In this study, the biomass of green algae U. intestinalis was subjected to four pretreatment techniques O 3 , US, MW and Fe 3 O 4 NPs, either individually or in combination at different doses, to increase its digestibility for processing biogas. The synthesis of green Fe 3 O 4 was confirmed by various characterization techniques such as TEM, SEM, FTIR and XRD. As a result, in contrast with the untreated biomass studies, the lowest dosages of US, O 3 (10 min), MW (2 min), and Fe 3 O 4 (5 mg/L) resulted in the highest biogas yield when sediment is used as a source of anaerobic bacteria. US efficacy in enhancing the solubility of organic matter to increase biogas generation from anaerobic digestion processes or accelerate the digestion of organic matter with reduced time, frequencies and power (10 min-50 Hz and 500 W) has been demonstrated in experimental activities. The findings also suggest that US pretreatment could be useful for lowering digest treatment expenses and increasing biogas generation. According to the energy study, combining MW pretreatment with Fe 3 O 4 NPs produced more energy while using less input energy than MW pretreatment alone. For each experimental scenario, the kinetic parameters of the reaction were scientifically analyzed using a modified Gompertz function model. The group with the MW pretreatment and Fe 3 O 4 NPs had a higher potential and maximal biogas generation rate. The shape of the modified Gompertz function model curves indicated that the majority of both the experimental and predicted biogas data was well fitted and confirmed by R 2 and RSME.