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Article

Exploring Optimal Pretreatment Approaches for Enhancing Biohydrogen and Biochar Production from Azolla filiculoides Biomass

by
Mohamed El-Qelish
1,2,
Saber A. El-Shafai
1 and
Mohamed Mahmoud
1,3,*
1
Water Pollution Research Department, National Research Centre, Cairo 12311, Egypt
2
Chemical and Biochemical Engineering Department, University of Western Ontario, London, ON N6A 5B9, Canada
3
Faculty of Engineering, Galala University, Galala City 43511, Suez, Egypt
*
Author to whom correspondence should be addressed.
Water 2024, 16(21), 3048; https://doi.org/10.3390/w16213048
Submission received: 14 September 2024 / Revised: 21 October 2024 / Accepted: 22 October 2024 / Published: 24 October 2024
(This article belongs to the Special Issue Biological Treatment of Water Contaminants: A New Insight)
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Abstract

:
Mitigating the irreversible consequences of climate change necessitates the application of sustainable energy resources. Hereby, we investigated the biological anaerobic fermentation of Azolla filiculoides biomass for biohydrogen production as a clean renewable energy source. Azolla filiculoides is a widely growing aquatic plant in polluted freshwater streams. However, the high non-biodegradable organic matter content in Azolla filiculoides biomass remains challenging in efficiently producing renewable energy, especially when it is being used as the sole donor substrate. In order to overcome this challenge, different pretreatment scenarios (namely, alkali, autoclaving, and ultrasonication) have been employed for enhancing the hydrolysis of Azolla filiculoides biomass to maximize the anaerobic fermentation and, consequently, the biohydrogen production potential. The biohydrogen production potential was 250.5, 398, 414.5, and 439.5 mL-H2, giving a hydrogen yield of 60.1, 89.6, 92.9, and 107.9 mL-H2/g-VS, respectively. Gompertz kinetics were applied to estimate the growth parameters of the process, which revealed a good fit with R2 ranging from 0.96 to 0.98. The produced digestate was valorized for biochar production, a material that could be applied for water treatment purposes. The produced biochar was characterized using different physical analyses, including FTIR, SEM, EDX, and TEM. The physicochemical characterizations of biochar demonstrate a successful formation of biochar with a highly porous structure and a rough surface, as evidenced by the emergence of significant functional groups (e.g., O-H, C-H, C=C, and C=O) existing on the surface of the biochar. In conclusion, this study harnesses a sustainable approach for the treatment of organic waste streams, which represents a circular economy model by transforming waste materials into valuable products and reducing the reliance on non-renewable resources.

1. Introduction

The rapid growth of the global economy has led to a surge in fossil fuel consumption, resulting in energy resource depletion and enormous greenhouse gas emissions. To address these challenges, biofuels, such as biohydrogen, bioethanol, biomethane, and biodiesel, represent promising, environmentally friendly alternatives to fossil fuels. In this context, biohydrogen represents a clean energy source owing to its high energy content and zero-carbon emissions [1,2]. Biohydrogen can be produced through several biochemical approaches, including anaerobic fermentation (dark- and photo-fermentation), biophotolysis (direct and indirect), and bioelectrochemical technologies [3,4,5]. Among the potential technologies for biohydrogen production, the anaerobic dark fermentation process (ADFP), which is a fast-growing technology for biohydrogen production from waste streams, is known for its high performance [6,7]. However, the process instability and sluggish kinetics of biochemical reactions are the main challenges that restrict its commercialization and scaling up [8,9]. ADFP occurs through two main consecutive phases, hydrolysis and acidogenesis, which are catalyzed by different microbial species. The hydrolysis of complex substrates into simpler forms represents the rate-limiting step of the ADFP, which if accelerated would increase biohydrogen production and enhance the overall process efficiency [10,11].
The water fern or Azolla filiculoides is naturally present in polluted freshwater bodies and can be efficiently employed to remove a wide range of pollutants in wastewater, with the subsequent utilization of plant biomass for renewable energy and petrochemical production [12,13]. Given the high organic matter content (e.g., proteins and amino acids) and low lignin content (i.e., <3%), aquatic macrophytes, including Azolla filiculoides, represent a potential feedstock for dark fermentative biohydrogen production [14]. In addition, the accessible harvesting of aquatic macrophytes from natural water bodies and their subsequent application as feedstock in the anaerobic fermentation process has many environmental benefits, including enhancing the natural purification capacity of the water bodies by removing excess biomass and providing more space for the residual mates to grow effectively and remove more pollutants [15]. This also prevents overcrowding and competition, which declines the natural purification capacity and causes aging and decay of the lower plant mate due to light shading. A recent study revealed that Azolla filiculoides, growing on municipal sewage as the sole donor substrate, has a significantly higher biomass yield (1.3-ton h−1) compared with duckweed (0.9-ton h−1) with a comparable protein content [13].
The dark fermentative hydrogen production process often proceeds at a relatively low hydraulic retention time, making utilizing complex solid waste streams as the sole donor substrate challenging. Although hydrolysis and fermentation can occur together in the same bioreactor, it may be beneficial to arrange for part of the sluggish hydrolysis to take place in a separate reactor before the ADFP bioreactor. Over the past few decades, several pretreatment strategies have been proposed to facilitate the hydrolysis phase of complex solid waste streams in order to provide simple substrates for hydrogen-producing fermenters [16]. The overarching goal of the pretreatment step, including thermal, chemical, and irradiation, is to increase the availability of fermentable substrates for microorganisms by breaking the cell walls and releasing the fermentable substrates, thus increasing the hydrogen yield [17]. For instance, alkaline pretreatment was shown to be more efficient than acidic pretreatment in enhancing the solubilization of proteins [18], which comprise a significant fraction of the organic material of Azolla filiculoides (i.e., 25.6%) [19].
Despite the improvement in substrate utilization efficiency, effluents from the dark fermentative hydrogen production process still contain significant amounts of chemical oxygen demand (COD) and nutrients, which if not properly treated would result in serious environmental issues, such as eutrophication [20]. For example, anaerobic fermentation digestates, which are a by-product of the dark fermentation of waste streams, have been used as a feedstock for biochar production via pyrolysis at a temperature range of 300–900 °C, which can be beneficially used as a promising adsorbent and potential soil amendment [21,22]. Furthermore, digestate-based biochar can improve soil quality by increasing its cation-exchange capacity and reducing the leaching of nitrogen and other nutrients into the groundwater [23]. The application of biochar as a fertilizer for soil amendment depends on the feedstock and production conditions [24], while its application of bioremediation depends on the physical properties, including surface morphology, surface area, pore volume/pore size distribution, and functional groups [25]. Although there has been limited research on reusing solid digestate as a biomass precursor to produce biochar, most of the published studies have focused on preparing biochar at relatively low temperatures.
In this study, we investigated biohydrogen production during the anaerobic dark fermentation of Azolla filiculoides biomass, which is a widely growing aquatic macrophyte in polluted freshwater streams, as a clean source of energy. However, the high non-biodegradable content in Azolla filiculoides biomass remains challenging for efficiently producing renewable energy, especially when it is being used as the sole donor substrate. In order to overcome this challenge, we employed different pretreatment scenarios (i.e., alkali, autoclaving, and ultrasonication) to improve biomass hydrolysis and maximize the biohydrogen production potential [26]. In addition, we explored the feasibility of utilizing the digestate produced during the anaerobic dark fermentative process of Azolla filiculoides for the production of biochar, which was characterized by different analytical tools, such as Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDX), and transmission electron microscopy (TEM).

2. Materials and Methods

2.1. Inoculum Sludge

Mixed-culture bacteria for assessing the biohydrogen production potential (BHP) was collected from the returned waste activated sludge line of the Gabal Al-Asfer wastewater treatment facility (Cairo, Egypt). After collection, the sludge samples were settled down for 2 h and the supernatant was discarded. Hydrogen producers were harvested by treating the sludge at 100 °C for 30 min as described elsewhere [18]. The heat-treated sludge is called inoculum hereafter. The pH, total solids (TS), and volatile solids (VS) of the inoculum were 7.29, 6.50 g/L, and 4.55 g/L, respectively.

2.2. Azolla filiculoides Sampling and Pretreatment Scenarios

Azolla filiculoides was sampled using a plastic net during the winter of 2023, from a small irrigation canal in Giza Governorate, Egypt. After collection, snails and other debris were manually removed, and the samples were thoroughly washed with Milli-Q water, dried at room temperature for one day, and then dried in an oven at 105 °C overnight. The resultant dried Azolla filiculoides was ground into a powder using an electrical mixer and sieved using a 0.6 mm stainless-steel sieve (Blau-Metall Inh., Triptis, Germany). Azolla filiculoides powder (AP) had a TS, VS, total Kjeldahl nitrogen (TKN), and total phosphorous (TP) of 4.8%, 3.8%, 48.3 mg/100 mg, and 3.0 mg/100 mg, respectively.
AP was hydrolyzed using three pretreatment approaches: alkali, ultrasonication (US), and thermal autoclaving. In all the pretreatment steps, 5 g of AP was suspended in 50 mL of Milli-Q water. The alkali pretreatment was accomplished by increasing the pH of AP to 12.0 under continuous stirring using 10 M NaOH, followed by static incubation at room temperature for 24 h. The ultrasonication pretreatment was performed using probe-type ultrasonic processing equipment with a probe diameter of 19 mm (Sonics & Materials Inc., Newtown, CT, USA). AP was ultrasonicated for 30 min at a frequency of 20 kHz. The thermal autoclaving was performed by heating the sample at 121 °C under a pressure of 1.5 bars for 15 min in a Purister 80 laboratory autoclave (Cryste, Gyeonggi-do, Republic of Korea).

2.3. Batch Biohydrogen Production Assays

The biohydrogen production potential (BHP) of pretreated AP was conducted in 130 mL serum glass bottles with an effective working volume of 75 mL. Each bottle was inoculated with 20 mL of heat-treated inoculum and 55 mL of pretreated AP solution. A blank set was performed by incubating serum bottles with 20 mL of inoculum and 55 mL of Milli-Q water. The initial pH of all the batch assays was adjusted to 6.5 using 4.0 M NaOH or 5.0 M HCl. After inoculation, all the bottles were purged with ultrapure nitrogen gas for 3 min to eliminate any oxygen and provide an anaerobic condition for the anaerobic fermenters, then they were quickly closed with rubber septum stoppers (20 mm, Bellco Glass Inc., Vineland, NJ, USA), and sealed with aluminum lids. All the batch assays were performed in a shaking incubator (120 rpm) at a fixed temperature of 37 ± 1 °C. The generated overpressure due to gas expansion, when the temperature was increased from room temperature to 37 °C, was released two hours after incubation to avoid a false gas measurement due to interference with the actual gas production because of bioactivity. All the batch experiments were conducted in triplicates.

2.4. Analysis and Calculations

Quantitative and qualitative analyses of the produced biogas (H2, CH4, and CO2) were carried out using the syringe method as described elsewhere [27] using gas chromatography (GC Agilent 7890, Agilent Technologies, Santa Clara, CA, USA) equipped with a molecular sieve silica gel column and thermal conductivity detector as described elsewhere [28]. A physicochemical analysis for COD, TKN, TS, VS, and TP was measured according to the standard method for the analysis of water and wastewater [29]. Soluble fractions were determined after filtering the samples through a 0.45 µm membrane filter. Fermentation by-products (i.e., formic acid (HFo), lactic acid (HLa), acetic acid (Hac), propionic acid (HPr), and butyric acid (HBu)) were quantified using high-performance liquid chromatography (Thermo Scientific, Germering, Germany) equipped with an Aminex HPX-87H column [30]. Before injection, the samples were centrifuged at 10,000 rpm for 10 min and filtered on a 0.2 μm membrane filter (Thermo Fisher Scientific, Germany). Lignin, cellulose, and hemicellulose were characterized according to National Renewable Energy Laboratory (NREL) methods [31].
A modified Gompertz equation was used to investigate the BHP kinetics according to the following equation:
H t = P × exp   − exp R m e p     λ − t   + 1    
where H represents the cumulative biohydrogen production (mL) at reaction time (t); P is the biohydrogen production potential; Rm is the maximum rate of biohydrogen formation (mL-H2 h −1); and λ is the duration of the lag phase (days) [32]. The significant difference between the studied pretreatments was assessed using an IBM SPSS statistical analysis (ANOVA) and the Tukey test, considering results to be statistically significant at p < 0.05 [33].

2.5. Preparation and Characterization of Biochar Produced from BHP Digestate

The digestate at the end of the batch BHP assays was filtered through a qualitative filter paper and the suspended matter was dried at 45 °C for 24 h. The dried digestate was thereafter ground into the desired powder form. Following the drying stage, the dried digestate powder was pyrolyzed in a muffle furnace at 600 ± 1 °C for 4 h with a heating rate of 10 °C/min and a nitrogen flow rate of 50 mL/min [30,34]. Finally, the obtained biochar powder was washed several times with Milli-Q water and dried at 105 °C overnight, which was followed by grinding in a ball-milling container (Photon Scientific Model: ph-BML912) for 5 h at 600 rpm.
The functional and structural composition of the produced biochar was assessed using Fourier-transform infrared spectroscopy (FTIR) (Shimadzu FTIR 8101 PC infrared spectrophotometer, Tokyo, Japan) at a scanning range of 4000 to 400 cm−1. Scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) were employed using a high-resolution Quanta FEG 250 instrument (FEI, Eindhoven, The Netherlands) at 20 kV to detect the morphological and elemental composition of the prepared digestate biochar. Before SEM imaging, samples were mounted on aluminum stubs using carbon tape, and then coated with a thin layer of gold (Au) for 90 s using a Q150t sputter coater (The Quorum Techniques Ltd., East Sussex, UK).

3. Results and Discussion

3.1. Effect of Different Pretreatment Approaches for Enhancing Hydrolysis of Azolla filiculoides

In this study, we studied the impact of three different pretreatments (i.e., alkali, autoclaving, and US) on AP, aiming to improve the hydrolysis of particulate matter into more readily biodegradable compounds towards enhancing the biohydrogen yield. Figure 1 demonstrates a ~1.6- to 2-fold increase in the soluble COD (SCOD) concentration upon applying pretreatment strategies, with ultrasonication pretreatment being the most effective pretreatment as evidenced by the increasing SCOD from 5.95 ± 0.11 g/L (in the control bioreactor) to 11.72 ± 0.18 g/L. The high COD solubilization is mainly attributed to the conversion of the particulate COD (PCOD), of which lignin, cellulose, and hemicellulose represent 36% (Table 1). Lignin commonly serves as a protective barrier around cellulose and hemicellulose, blocking hydrolyzing enzymes from accessing these readily biodegradable components. Therefore, the applied pretreatment strategies destroyed the lignin structure, expanded the surface area, and decreased the cellulose crystallinity [1]. All the pretreatment strategies achieved a remarkably high degree of lignin destruction, with US pretreatment exhibiting the highest destruction efficiency of 34.52%.
Our results are in agreement with previous studies, demonstrating that pretreatment strategies resulted in the hydrolysis and breakdown of hardly biodegradable organic matter, such as cellulose and hemicellulose, into simpler organic substrates, which can be efficiently utilized by hydrogen-producing fermenters for maximizing the energy output [35,36]. For instance, alkali pretreatment, which is a widely applied pretreatment method for increasing the hydrolysis rate of lignocellulosic biomass through nucleophilic substitution reactions, where the metal ion acts as a nucleophile, attacks the lignocellulosic biomass structure, and creates new covalent bonds by destroying the original bonds resulting in the hydrolysis of the lignocellulosic biomass [37]. Lignin, cellulose, and hemicellulose removal efficiency through the anaerobic digestion process increased by 9.3–19.1% with NaOH pretreatment [38], while the application of CaO alkali pretreatment improved the lignin removal efficiency by 25% [39]. The biogas potential of dairy cow manure increased by 23.6% with alkali pretreatment (10% NaOH combined with thermal pretreatment at 100 °C for 5 min) [40], while a 78% increase was observed in the anaerobic digestion of pig manure under alkali pretreatment at pH 10 [41], which was attributed to the release of soluble COD into the liquid phase. Thermal hydrolysis is also commonly used as a pretreatment technology for various biowaste materials due to its simplicity, applicability, and efficiency. The effectiveness of thermal hydrolysis relies on breaking down large macromolecular biopolymers into simpler, soluble molecules, making them more accessible for bacterial enzymatic action [42]. Additionally, thermal hydrolysis can disrupt the gel structure of the AP, releasing organic substances trapped within the cell membrane into the liquid phase. Different temperatures (50–160 °C) and reaction times (from several minutes to several hours) have been tested to optimize the conditions for each type of biomass [42,43,44]. For instance, Ray et al. [45] reported that the autoclaving pretreatment yielded the most efficient results, as confirmed by both the morphological and chemical properties of the pretreated sample, where the pretreated microalgal biomass sample showed the lowest crystalline index of 2.1% and the highest hydrogen production of 1.543 L/L. In another study, Passaro et al. [46] pretreated sewage sludge using the US at a low frequency of 24 kHz and a density of 0.8 W/mL and reported that the higher the energy input released, the higher the SCOD concentrations. The COD solubilization was attributed to the release of extracellular polymeric substances (EPSs) from the disintegration of the floc structure and the release of intracellular organic substances from cell lysis [47].

3.2. Effect of Different Pretreatments on BHP

Figure 2 shows the cumulative hydrogen production of the anaerobic fermentation of the Azolla filiculoides substrate with different pretreatment strategies. Our results revealed that cumulative hydrogen production was remarkably increased upon applying different pretreatment strategies. The ultrasonicated pretreated Azolla filiculoides substrate exhibited a cumulative hydrogen production of 439.5 mL-H2—~1.1–1.8-fold higher than that of other anaerobic bioassays (i.e., 250.5 mL-H2 for the control Azolla filiculoides substrate, 398 mL-H2 for the alkali pretreated Azolla filiculoides, and 414.5 mL-H2 for the autoclaved pretreated Azolla filiculoides), giving a hydrogen yield of 60.1, 89.6, 92.9, and 107.9 mL-H2/g-VS for the control Azolla filiculoides, alkali pretreated AP, autoclaved pretreated AP, and ultrasonicated pretreated AP, respectively. The maximum hydrogen production rate (Rmax), which refers to the peak rate of hydrogen production during the process, was estimated to be 56.0, 54.5, 62.5, and 67.5 mL-H2/day for the control, alkali, autoclaving, and US pretreated Azolla filiculoides, respectively. More interestingly, we observed that approximately 75% of the H2 was produced within 7 days of the total incubation period of 25 days, which is a critical economical assessment parameter of the process. The high concentration of simple sugars resulting from the pretreatment methods of the waste, combined with the presence of hydrogen-producing bacteria introduced through the sludge heat-shock pretreatment, enhanced hydrogen production in the process in the first days of the anaerobic fermentation process. The three applied pretreatments increased the BHP and hydrogen yield by 58.9, 65.5, and 75.4% for the alkali, autoclaving, and US, respectively (Table 2). The anaerobic fermentation process is the fastest phase of the anaerobic digestion process, which includes two main steps, namely hydrolysis and acidogenesis [48]. This phase takes place at a low hydraulic retention time (from several hours to a maximum of two days) and a high organic loading rate. These experimental conditions should be adapted for the hydrogen-producing bacteria to prevent the growth of the hydrogen-consuming bacteria and other substrate competitors [49]. The different applied pretreatments facilitated the hydrolysis stage of the anaerobic fermentation, which is the rate-limiting step of this biochemical reaction by solubilizing the particulate organic matter into soluble compounds. Our results are in agreement with previous findings, demonstrating that H2 production from food waste was enhanced by applying alkali (pH 12 for 24 h) and US (30 min at a frequency of 20 kHz) pretreatment separately, giving a BHP of 228.5 mL-H2 (yield of 3.7 mL-H2/g-VS added) for the alkali and 285.0 mL-H2 (yield of 4.77 mL-H2/g-VS added) for the US compared with 195.5 mL-H2 (yield of 3.22 mL-H2/g-VS added) for the untreated control [50]. In another study, fruit and vegetable waste was autoclaved (at 105 °C for 10 min) before being anaerobically fermented for biohydrogen production and the autoclaving pretreatment achieved a hydrogen yield (27.19 mL H2/g-VS) that was 30.7% higher than the untreated control (20.81 mL H2/g-VS for control) [51]. Anaerobic fermentative hydrogen production from treated milk processing wastewater using US (25 kHz for 5 min), thermal (90 °C for 30 min), and chemical (pH 11 by adding Ca(OH)2) pretreatment increased the BHP by 14.84% (68.9 mL-H2), 170% (162 mL-H2), and 175.5% (165.3 mL-H2) [52].

3.3. Metabolites of the BHP of Azolla filiculoides

The anaerobic fermentation process involves two main biochemical stages, namely hydrolysis and acidogenesis. Hydrolysis is responsible for the conversion of complex compounds into simpler forms, where carbohydrates are hydrolyzed into mono- and di-sugars, proteins are hydrolyzed into amino acids, and peptides and lipids are hydrolyzed into long-chain fatty acids and triglycerides. Acidogenesis involves the conversion of the simpler substrate forms into different volatile fatty acids (VFAs) depending on the metabolic pathway. Acetate, propionate, butyrate, and isobutyrate were the main metabolites in the anaerobic fermentation of AP (Figure 3). Hydrogen production through the acetate and butyrate pathways is the most common with a theoretical hydrogen yield of 4 and 2 mol of hydrogen per 1 mol of glucose, respectively [53,54]. However, the TVFA concentration was 19.67, 28.81, 15.6, and 19.70 g COD/L for the control, alkali, autoclaving, and US, respectively. VFA production is often accompanied by a pH decrease [55], where the pH was decreased from 6.5 to 6.33, 5.33, 5.85, and 5.86 for the control, alkali, autoclaving, and US, respectively. Formic acid was not produced in the untreated control batch assay, although it was produced in the three treated AP batch assays, which could be attributed to the high pH in the control assays due to the lower TVFA compared with the three treated assays. Formate production at pH was previously reported in the anaerobic fermentation of glucose using a mixed culture [56]. Similarly, Infantes et al. [57] observed a correlation between the formic acid concentration and pH, noting that formic acid levels were nearly zero at pH 4 and 5, and were only produced when the culture was maintained at pH 6. Caproic acid appeared in the alkali-pretreated AP batch assay at a concentration of 33.85 mM. Caproic acid production is likely due to the secondary fermentation of ethanol and acetate or butyrate by specific species such as Clostridium kluyveri [58], where caproate formation does not consume hydrogen but rather produces it. Valerate production is always accompanied by hydrogen consumption, where the reaction of propionic acid and hydrogen produces valerate according to the following equation: CH3CH2COOH + 6H2 → CH3(CH2)3COOH [59].

3.4. Digestate Valorization for Biochar Production

The produced biochar was subjected to intensive characterization to determine the physicochemical properties and assess its materiality in several applications. Generally, the biochar yield varies according to several factors, including pyrolysis temperature, retention time, heating rate, and biomass composition. In our experimental setup, we observed a relatively high biochar yield of 0.4 g per gram of digestate. In addition, Figure 4a–c shows the SEM imaging of the as-prepared biochar of the AP digestate at different magnifications. The as-prepared biochar exhibited a highly porous structure with a rough surface. The porous configuration enables the pyrolysis products to escape the bulk of the AP without affecting its structural integrity. In addition, this highly porous configuration allows gas and combustion products to circulate freely in and out of the biomass powder during the pyrolysis process [60]. The inorganic ash content of the as-prepared AP digestate biochar represented 5%, which resulted from the combustion in the presence of air and the mineral salts in the digestate. The EDX elemental analysis showed that carbon and oxygen are the main components of the biochar (i.e., 83.26% and 16.74%, respectively). However, minor elements were detected in the ash fraction, including sodium, potassium, calcium, phosphorous, and sulfur, which demonstrates the substantial capacity of the AP in the mineral’s uptake [61]. The presence of the inorganic elements in the biochar triggers its catalytic properties in the high-temperature decomposition reactions that take place in both inert and oxidative mediums; thus, decreasing the amount of heat required for reaction accomplishment. Inorganic metallic salts that exist in the biochar of the biomass can catalyze the decomposition of cellulose [60]. Elements, such as sodium, potassium, and calcium, can also catalyze secondary reactions between CO2 in the pyrolysis gas and the biochar [62].
The FTIR spectrum of the AP biochar demonstrates the emergence of significant functional groups existing on the surface of the biochar in the range between 4000 and 400 cm−1. We detected a broad O-H stretching band at 3374 cm−1 and weak aliphatic C-H stretching vibrations at 2921 cm−1, indicating the presence of heat-resistant aliphatic structures in the as-prepared biochar. The sharp peak at 1650 cm−1 is characteristic of the aromatic carbon including the C=C vibrations and C=O, which originates from the polysaccharide skeleton that exists in the cellulose and hemicellulose of the AP [63]. The stretching vibration at 1040 cm−1 could be assigned to the metal oxides [64]. The small bands that appear between 800 and 600 cm−1 are distinctive of the inorganic ashes and salts. The AP digestate biochar is rich in functional groups, which could enhance its cation exchange properties, opening the way for its application as an efficient biosorbent in wastewater treatment [30].

3.5. Outlook

Achieving the circular economy concept requires the reduction of waste streams, while recovering valuable materials. This was originally developed to replace the traditional linear economy, which results in the production of several valuable products and secondary waste materials, requiring additional treatments and disposal steps. The basis of the circular economy relies on the fundamentals of environmental economics and environmental sustainability [65]. Transitioning to a circular economy requires balancing industrial and economic growth with environmental protection and efficient resource use. Since the onset of the Industrial Revolution, most global economies have relied heavily on linear practices (consume, waste, and dispose of), which has resulted in the overconsumption of resources without wise planning for resource recovery and reuse. Hereby, Azolla filiculoides is a widespread aquatic plant that represents an enormous source of biomass for green energy production through the anaerobic dark fermentation process, which is comparable with data from the previous literature (Table 3). However, the digestate produced during the dark fermentation of Azolla filiculoides has high VFAs and biomass. The VFAs in the liquid digestate could be separated and used in many industries, including cosmetics, beverages, food, medicinal, and chemical industries [7]. In addition, the solid fraction of the digestate could be used in biochar production, which is another value-added product. Biochar’s unique characteristics, including the high specific surface area, high porosity, catalytic abilities, high conductivity, and stability, make it highly effective as a biosorbent for various pollutants, including heavy metals, dyes, and organic contaminants. Additionally, the presence of valuable elements, such as phosphorus, nitrogen, sodium, calcium, and potassium, in biochar contributes to its use as a soil amendment. The produced biochar demonstrated a moderate carbon sequestration potential when added to the soil, with an estimated half-life ranging from 100 to 1000 years [66].

4. Conclusions

Our substantial reliance on non-renewable fossil fuels to supply global energy requirements has led to an unprecedented rise in global greenhouse gas (GHG) emissions, including carbon dioxide. Thus, satisfying our increasing energy demand using sustainable renewable energy sources appears promising to achieve carbon neutrality; however, the exact technologies that will allow us to transition from utilizing fossil fuels to carbon-neutral energy sources are still unknown. In this study, we investigated the feasibility of biohydrogen production during the anaerobic dark fermentation of Azolla filiculoides biomass, which is a widely growing aquatic plant in polluted freshwater streams, as a clean source of energy. In order to improve the substrate availability, we employed different pretreatment scenarios (i.e., alkali, autoclaving, and ultrasonication) to improve biomass hydrolysis and maximize the biohydrogen production potential. The biohydrogen production potential was 250.5, 398, 414.5, and 439.5 mL-H2, giving a hydrogen yield of 60.1, 89.6, 92.9, and 107.9 mL-H2/g-VS, respectively. We also investigated the valorization of digestate produced during the anaerobic digestion of Azolla filiculoides in biochar production, which could be applied in various applications, including water and wastewater purification. The physicochemical characterizations of biochar demonstrate a successful formation of biochar with a highly porous structure and a rough surface. Furthermore, FTIR showed the emergence of significant functional groups (e.g., O-H, C-H, C=C, and C=O) existing on the surface of the biochar. Our study provides insight into valuable resource recovery (i.e., renewable biohydrogen and biochar) from aquatic macrophytes, which represent a serious pollution source in freshwater streams. This approach embodies a circular economy paradigm by converting waste materials into useful products and decreasing the dependence on non-renewable resources.

Author Contributions

M.E.-Q., S.A.E.-S. and M.M. conceived and designed the experiments. M.E.-Q. performed the experiments. M.E.-Q., S.A.E.-S. and M.M. analyzed the data. M.E.-Q. wrote the first draft of the paper. S.A.E.-S. and M.M. critically revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The dataset is available upon request from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 16 03048 g001
Figure 1. Effect of different pretreatments on the Azolla filiculoides substrate COD solubilization.
Figure 1. Effect of different pretreatments on the Azolla filiculoides substrate COD solubilization.
Water 16 03048 g001
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Figure 2. Hydrogen production potential (a) and yield of the Azolla filiculoides substrate (b) with different pretreatment strategies in mesophilic conditions.
Figure 2. Hydrogen production potential (a) and yield of the Azolla filiculoides substrate (b) with different pretreatment strategies in mesophilic conditions.
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Figure 3. Metabolites produced during the anaerobic fermentation of the Azolla filiculoides substrate with different pretreatment strategies.
Figure 3. Metabolites produced during the anaerobic fermentation of the Azolla filiculoides substrate with different pretreatment strategies.
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Figure 4. SEM (a–c), EDX (d), and FTIR (e) of biochar prepared from the Azolla filiculoides anaerobic fermentation digestate using pyrolysis at 600 °C for 4 h.
Figure 4. SEM (a–c), EDX (d), and FTIR (e) of biochar prepared from the Azolla filiculoides anaerobic fermentation digestate using pyrolysis at 600 °C for 4 h.
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Table 1. Summary of the anaerobic digesters’ efficiency in treating different pretreated Azolla filiculoides substrates.
Table 1. Summary of the anaerobic digesters’ efficiency in treating different pretreated Azolla filiculoides substrates.
ParameterControlAlkaliAutoclavingUltrasonication
TCOD (g/L)61.20 ± 1.361.90 ± 1.4562.40 ± 1.5562.00 ± 1.10
SCOD (g/L)5.95 ± 0.119.50 ± 0.199.96 ± 0.2111.72 ± 0.18
PCOD (g/L)55.25 ± 1.1952.41 ± 1.2652.44 ± 1.3450.28 ± 0.92
COD solubilization-59.5867.3996.97
Lignin (g/L)5.884.504.103.85
Lignin removal (%)-23.4730.2734.52
Cellulose (g/L)11.908.438.017.36
Cellulose removal (%)-29.1632.6938.15
Hemicellulose (g/L)2.101.501.101.10
Hemicellulose removal (%)-28.5747.6247.62
Table 2. Gompertz equation parameters fitted to the experimental data of the anaerobic fermentation of pretreated Azolla filiculoides substrates at an initial pH of 6.5 and 37 °C.
Table 2. Gompertz equation parameters fitted to the experimental data of the anaerobic fermentation of pretreated Azolla filiculoides substrates at an initial pH of 6.5 and 37 °C.
Gompertz ParametersControlAlkaliAutoclavingUltrasonication
P (mL-H2)250.50398.00414.50439.50
Rmax (mL-H2/day)56.0054.5062.5067.50
λ (d)1.001.001.001.00
R20.990.990.990.99
Yield (mL-H2/g-TS)50.179.682.987.9
H2 increase (%)-58.965.575.4
Table 3. Hydrogen yield from the anaerobic fermentation of different aquatic plants pretreated with different pretreatment conditions and operated at different operational conditions.
Table 3. Hydrogen yield from the anaerobic fermentation of different aquatic plants pretreated with different pretreatment conditions and operated at different operational conditions.
SubstrateInoculumOperational ConditionsSubstrate PretreatmentYieldReference
Dried biomass of Azolla filiculoidesEnterobacter cloacae DT-1Batch
2 L reactor
Initial pH 7.5
37 °C		1% H2SO4 + autoclaving at 120 °C for 60 min2.43 mol H2/mol substrate[19]
Dried biomass of Azolla filiculoidesEnterobacter cloacae DT-1Batch
2 L reactor
Initial pH 7.5
37 °C		Enzymatic saccharification at pH 5 and
50 °C for 24 h		2.04 mol H2/mol substrate[19]
Azolla pinnataPretreated anaerobic inoculum40 L reactor
Initial pH 7.0
25 °C		Acid treatment
2% HCl at 121 °C		14.6 mL-H2/g[67]
Eichhornia crassipesPretreated bird manure4.0 L reactor
Initial pH 6.0
30 °C		3% (v/v) H2SO4 at 60° C for 12 h81.3 mL-H2/g[68]
Duckweed biomassPretreated activated sludgeBatch
35 °C
pH 7.7		Autoclave at 121 °C, 15 psi for 30 min144.04 mL H2/g COD removed[69]
Duckweed biomassPretreated activated sludgeBatch
50 °C
pH 7.7		Autoclave at 121 °C, 15 psi for 30 min381.98 mL H2/g COD removed[70]
Duckweed biomassPretreated activated sludgeBatch
55 °C
pH 7.7		Autoclave at 121 °C, 15 psi for 30 min416.09 mL H2/g
COD removed		[70]
DuckweedPretreated anaerobic activated sludgeBatch
35 °C
pH of 7.0		1% (w/v) H2SO4 +
120 °C for 30 min		169.30 mL-H2/g[71]
DuckweedPretreated anaerobic activated sludgeBatch
35 °C
pH of 7.0		1% (w/v) NaOH +
120 °C for 30 min		121 mL-H2/g[71]
DuckweedPretreated anaerobic activated sludgeBatch
35 °C
pH of 7.0		120 °C for 30 min60 mL-H2/g[71]
Azolla microphyllaEnterobacter cloacae DT-1Batch
pH 7.5
37 °C		Enzymatic hydrolysis2.1 mole-H2/gVS[72]
water hyacinth
(Eichhornia crassipes)		Enterobacter cloacae DT-1Batch
pH 7.5
37 °C		Enzymatic hydrolysis1.42 mole-H2/gVS[72]
Spirulina sp.Enterobacter cloacae DT-1Batch
pH 7.5
37 °C		Enzymatic hydrolysis2.16 mole-H2/gVS[72]
Scenedesmus sp.Enterobacter cloacae DT-1Batch
pH 7.5
37 °C		Enzymatic hydrolysis2.0 mole-H2/gVS[72]
Eichhornia crassipesEnterobacter cloacae DT-1Batch
pH 7.5
37 °C		Acid hydrolysis—diluted H2SO425 mmol-H2/L[73]
Azolla microphyllaEnterobacter cloacae DT-1Batch
pH 7.5
37 °C		Acid hydrolysis—diluted H2SO412 mmol-H2/L[73]
Alternanthera philoxeroidesEnterobacter aerogenes ZJU1pH 6.0
37 °C		1.0 v/v% H2SO4, 135 °C for 15 min62.2 mL-H2/gVS[74]
Pistia stratiotesPretreated anaerobic activated sludgeBatch
pH 5.5
25 °C		1% (w/v) of 2.5 H2SO4 for 45 min2.3 mol-H2/mol-glucose[74]
Azolla filiculoidesPretreated anaerobic activated sludgeBatch
pH 6.5
37 °C		Ultrasonication107.9 mL-H2/gVSThis study
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El-Qelish, M.; El-Shafai, S.A.; Mahmoud, M. Exploring Optimal Pretreatment Approaches for Enhancing Biohydrogen and Biochar Production from Azolla filiculoides Biomass. Water 2024, 16, 3048. https://doi.org/10.3390/w16213048

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El-Qelish M, El-Shafai SA, Mahmoud M. Exploring Optimal Pretreatment Approaches for Enhancing Biohydrogen and Biochar Production from Azolla filiculoides Biomass. Water. 2024; 16(21):3048. https://doi.org/10.3390/w16213048

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El-Qelish, Mohamed, Saber A. El-Shafai, and Mohamed Mahmoud. 2024. "Exploring Optimal Pretreatment Approaches for Enhancing Biohydrogen and Biochar Production from Azolla filiculoides Biomass" Water 16, no. 21: 3048. https://doi.org/10.3390/w16213048

APA Style

El-Qelish, M., El-Shafai, S. A., & Mahmoud, M. (2024). Exploring Optimal Pretreatment Approaches for Enhancing Biohydrogen and Biochar Production from Azolla filiculoides Biomass. Water, 16(21), 3048. https://doi.org/10.3390/w16213048

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